AAV-mediated neuronal expression of an scFv antibody selective for Aß oligomers protects synapses and rescues memory in Alzheimer models.

The accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) in the brain has been implicated in synapse failure and memory impairment in Alzheimer's disease. Here, we initially show that treatment with NUsc1, a single-chain variable-fragment antibody (scFv) that selectively targets a subpopulation of AßOs and shows minimal reactivity to Aß monomers and fibrils, prevents the inhibition of long-term potentiation in hippocampal slices and memory impairment induced by AßOs in mice. As a therapeutic approach for intracerebral antibody delivery, we developed an adeno-associated virus vector to drive neuronal expression of NUsc1 (AAV-NUsc1) within the brain. Transduction by AAV-NUsc1 induced NUsc1 expression and secretion in adult human brain slices and inhibited AßO binding to neurons and AßO-induced loss of dendritic spines in primary rat hippocampal cultures. Treatment of mice with AAV-NUsc1 prevented memory impairment induced by AßOs and, remarkably, reversed memory deficits in aged APPswe/PS1ΔE9 Alzheimer's disease model mice. These results support the feasibility of immunotherapy using viral vector-mediated gene delivery of NUsc1 or other AßO-specific single-chain antibodies as a potential therapeutic approach in Alzheimer's disease.

Differences in learning and memory between middle-aged female and male rats.

We observed differences in cognitive functions between middle-aged female and male Wistar rats. Both (like youngsters) discriminated new versus familiar objects, showing similar short- and long-term memory (STM and LTM, respectively). Only females show robust LTM for new location of an object. Both successfully form LTM of inhibitory avoidance, though males appeared to be amnesic for memory persistence. Habituation, locomotion, horizontal exploration, "stereotypies," fear, and anxiety-like behavior were similar for both, while vertical exploration was significantly higher in middle-aged and younger females. Therefore, sex-dependent differences in some cognitive functions and behaviors must be considered when designing and interpreting learning and memory studies.

Efficacy of a Nasal Spray Containing Iota-Carrageenan in the Postexposure Prophylaxis of COVID-19 in Hospital Personnel Dedicated to Patients Care with COVID-19 Disease.

BACKGROUND: Iota-Carrageenan (I-C) is a sulfate polysaccharide synthesized by red algae, with demonstrated antiviral activity and clinical efficacy as nasal spray in the treatment of common cold. In vitro, I-C inhibits SARS-CoV-2 infection in cell culture. RESEARCH QUESTION: Can a nasal spray with Iota-Carrageenan be useful in the prophylaxis of COVID-19 in health care workers managing patients with COVID-19 disease? STUDY DESIGN AND METHODS: This is a pilot pragmatic multicenter, randomized, double-blind, placebo-controlled study assessing the use of a nasal spray containing I-C in the prophylaxis of COVID-19 in hospital personnel dedicated to care of COVID-19 patients. Clinically healthy physicians, nurses, kinesiologists and other health care providers managing patients hospitalized for COVID-19 were assigned in a 1:1 ratio to receive four daily doses of I-C spray or placebo for 21 days. The primary end point was clinical COVID-19, as confirmed by reverse transcriptase polymerase chain reaction testing, over a period of 21 days. The trial is registered at ClinicalTrials.gov (NCT04521322). RESULTS: A total of 394 individuals were randomly assigned to receive I-C or placebo. Both treatment groups had similar baseline characteristics. The incidence of COVID-19 differs significantly between subjects receiving the nasal spray with I-C (2 of 196 [1.0%]) and those receiving placebo (10 of 198 [5.0%]). Relative risk reduction: 79.8% (95% CI 5.3 to 95.4; p=0.03). Absolute risk reduction: 4% (95% CI 0.6 to 7.4). INTERPRETATION: In this pilot study a nasal spray with I-C showed significant efficacy in preventing COVID-19 in health care workers managing patients with COVID-19 disease. CLINICAL TRIALS REGISTRATION: NCT04521322.

Reduced Expression of Hippocampal GluN2A-NMDAR Increases Seizure Susceptibility and Causes Deficits in Contextual Memory.

N-methyl-D-aspartate receptors are heterotetramers composed of two GluN1 obligatory subunits and two regulatory subunits. In cognitive-related brain structures, GluN2A and GluN2B are the most abundant regulatory subunits, and their expression is subjected to tight regulation. During development, GluN2B expression is characteristic of immature synapses, whereas GluN2A is present in mature ones. This change in expression induces a shift in GluN2A/GluN2B ratio known as developmental switch. Moreover, modifications in this relationship have been associated with learning and memory, as well as different pathologies. In this work, we used a specific shRNA to induce a reduction in GluN2A expression after the developmental switch, both in vitro in primary cultured hippocampal neurons and in vivo in adult male Wistar rats. After in vitro characterization, we performed a cognitive profile and evaluated seizure susceptibility in vivo. Our in vitro results showed that the decrease in the expression of GluN2A changes GluN2A/GluN2B ratio without altering the expression of other regulatory subunits. Moreover, rats expressing the anti-GluN2A shRNA in vivo displayed an impaired contextual fear-conditioning memory. In addition, these animals showed increased seizure susceptibility, in terms of both time and intensity, which led us to conclude that deregulation in GluN2A expression at the hippocampus is associated with seizure susceptibility and learning-memory mechanisms.

Early Long-Term Memory Impairment and Changes in the Expression of Synaptic Plasticity-Associated Genes, in the McGill-R-Thy1-APP Rat Model of Alzheimer's-Like Brain Amyloidosis.

Accruing evidence supports the hypothesis that memory deficits in early Alzheimer Disease (AD) might be due to synaptic failure caused by accumulation of intracellular amyloid beta (Aß) oligomers, then secreted to the extracellular media. Transgenic mouse AD models provide valuable information on AD pathology. However, the failure to translate these findings to humans calls for models that better recapitulate the human pathology. McGill-R-Thy1-APP transgenic (Tg) rat expresses the human amyloid precursor protein (APP751) with the Swedish and Indiana mutations (of familial AD), leading to an AD-like slow-progressing brain amyloid pathology. Therefore, it offers a unique opportunity to investigate learning and memory abilities at early stages of AD, when Aß accumulation is restricted to the intracellular compartment, prior to plaque deposition. Our goal was to further investigate early deficits in memory, particularly long-term memory in McGill-R-Thy1-APP heterozygous (Tg+/-) rats. Short-term- and long-term habituation to an open field were preserved in 3-, 4-, and 6-month-old (Tg+/-). However, long-term memory of inhibitory avoidance to a foot-shock, novel object-recognition and social approaching behavior were seriously impaired in 4-month-old (Tg+/-) male rats, suggesting that they are unable to either consolidate and/or evoke such associative and discriminative memories with aversive, emotional and spatial components. The long-term memory deficits were accompanied by increased transcript levels of genes relevant to synaptic plasticity, learning and memory processing in the hippocampus, such as Grin2b, Dlg4, Camk2b, and Syn1. Our findings indicate that in addition to the previously well-documented deficits in learning and memory, McGill-R-Thy1-APP rats display particular long-term-memory deficits and deep social behavior alterations at pre-plaque early stages of the pathology. This highlights the importance of Aß oligomers and emphasizes the validity of the model to study AD-like early processes, with potentially predictive value.

Histidine-Rich Glycoprotein Inhibits HIV-1 Infection in a pH-Dependent Manner.

Histidine-rich glycoprotein (HRG) is an abundant plasma protein with a multidomain structure, allowing its interaction with many ligands, including phospholipids, plasminogen, fibrinogen, IgG antibodies, and heparan sulfate. HRG has been shown to regulate different biological responses, such as angiogenesis, coagulation, and fibrinolysis. Here, we found that HRG almost completely abrogated the infection of Ghost cells, Jurkat cells, CD4+ T cells, and macrophages by HIV-1 at a low pH (range, 6.5 to 5.5) but not at a neutral pH. HRG was shown to interact with the heparan sulfate expressed by target cells, inhibiting an early postbinding step associated with HIV-1 infection. More importantly, by acting on the viral particle itself, HRG induced a deleterious effect, which reduces viral infectivity. Because cervicovaginal secretions in healthy women show low pH values, even after semen deposition, our observations suggest that HRG might represent a constitutive defense mechanism in the vaginal mucosa. Of note, low pH also enabled HRG to inhibit the infection of HEp-2 cells and Vero cells by respiratory syncytial virus (RSV) and herpes simplex virus 2 (HSV-2), respectively, suggesting that HRG might display broad antiviral activity under acidic conditions.IMPORTANCE Vaginal intercourse represents a high-risk route for HIV-1 transmission. The efficiency of male-to-female HIV-1 transmission has been estimated to be 1 in every 1,000 episodes of sexual intercourse, reflecting the high degree of protection conferred by the genital mucosa. However, the contribution of different host factors to the protection against HIV-1 at mucosal surfaces remains poorly defined. Here, we report for the first time that acidic values of pH enable the plasma protein histidine-rich glycoprotein (HRG) to strongly inhibit HIV-1 infection. Because cervicovaginal secretions usually show low pH values, our observations suggest that HRG might represent a constitutive antiviral mechanism in the vaginal mucosa. Interestingly, infection by other viruses, such as respiratory syncytial virus and herpes simplex virus 2, was also markedly inhibited by HRG at low pH values, suggesting that extracellular acidosis enables HRG to display broad antiviral activity.

NMDA Receptor Subunits Change after Synaptic Plasticity Induction and Learning and Memory Acquisition.

NMDA ionotropic glutamate receptors (NMDARs) are crucial in activity-dependent synaptic changes and in learning and memory. NMDARs are composed of two GluN1 essential subunits and two regulatory subunits which define their pharmacological and physiological profile. In CNS structures involved in cognitive functions as the hippocampus and prefrontal cortex, GluN2A and GluN2B are major regulatory subunits; their expression is dynamic and tightly regulated, but little is known about specific changes after plasticity induction or memory acquisition. Data strongly suggest that following appropriate stimulation, there is a rapid increase in surface GluN2A-NMDAR at the postsynapses, attributed to lateral receptor mobilization from adjacent locations. Whenever synaptic plasticity is induced or memory is consolidated, more GluN2A-NMDARs are assembled likely using GluN2A from a local translation and GluN1 from local ER. Later on, NMDARs are mobilized from other pools, and there are de novo syntheses at the neuron soma. Changes in GluN1 or NMDAR levels induced by synaptic plasticity and by spatial memory formation seem to occur in different waves of NMDAR transport/expression/degradation, with a net increase at the postsynaptic side and a rise in expression at both the spine and neuronal soma. This review aims to put together that information and the proposed hypotheses.

GluN1 and GluN2A NMDA Receptor Subunits Increase in the Hippocampus during Memory Consolidation in the Rat.

It is widely accepted that NMDA receptors (NMDAR) are required for learning and memory formation, and for synaptic plasticity induction. We have previously shown that hippocampal GluN1 and GluN2A NMDAR subunits significantly increased following habituation of rats to an open field (OF), while GluN2B remained unchanged. Similar results were obtained after CA1-long-term potentiation (LTP) induction in rat hippocampal slices. Other studies have also shown NMDAR up regulation at earlier and later time points after LTP induction or learning acquisition. In this work, we have studied NMDAR subunits levels in the hippocampus and prefrontal cortex (PFC) after OF habituation and after object recognition (OR), to find out whether rising of NMDAR subunits is a general and structure-specific feature during memory formation. In 1, 2 and 3 month old rats there was an increase in hippocampal GluN1 and GluN2A, but not in GluN2B levels 70 min after OF habituation. This rise overlaps with early phase of memory consolidation, suggesting a putative relationship between them. The increases fell down to control levels 90 min after training. Similar results were obtained in the hippocampus of adult rats 70 min after OR training, without changes in PFC. Following OF test or OR discrimination phase, NMDAR subunits remained unchanged. Hence, rising of hippocampal GluN1 and GluN2A appears to be a general feature after novel "spatial/discrimination" memory acquisition. To start investigating the dynamics and possible mechanisms of these changes, we have studied hippocampal neuron cultures stimulated by KCl to induce plasticity. GluN1 and GluN2A increased both in dendrites and neuronal bodies, reaching a maximum 75 min later and returning to control levels at 90 min. Translation and/or transcription and mobilization differentially contribute to this rise in subunits in bodies and dendrites. Our results showed that the NMDAR subunits increase follows a similar time course both in vitro and in vivo. These changes happen in the hippocampus where a spatial representation of the environment is being formed making possible short term and long term memories (STM and LTM); appear to be structure-specific; are preserved along life; and could be related to synaptic tagging and/or to memory consolidation of new spatial/discrimination information.

Amnesia of inhibitory avoidance by scopolamine is overcome by previous open-field exposure.

The muscarinic cholinergic receptor (MAChR) blockade with scopolamine either extended or restricted to the hippocampus, before or after training in inhibitory avoidance (IA) caused anterograde or retrograde amnesia, respectively, in the rat, because there was no long-term memory (LTM) expression. Adult Wistar rats previously exposed to one or two open-field (OF) sessions of 3 min each (habituated), behaved as control animals after a weak though over-threshold training in IA. However, after OF exposure, IA LTM was formed and expressed in spite of an extensive or restricted to the hippocampus MAChR blockade. It was reported that during and after OF exposure and reexposure there was an increase in both hippocampal and cortical ACh release that would contribute to "prime the substrate," e.g., by lowering the synaptic threshold for plasticity, leading to LTM consolidation. In the frame of the "synaptic tagging and capture" hypothesis, plasticity-related proteins synthesized during/after the previous OF could facilitate synaptic plasticity for IA in the same structure. However, IA anterograde amnesia by hippocampal protein synthesis inhibition with anisomycin was also prevented by two OF exposures, strongly suggesting that there would be alternative interpretations for the role of protein synthesis in memory formation and that another structure could also be involved in this "OF effect."

The proinflammatory RAGE/NF-κB pathway is involved in neuronal damage and reactive gliosis in a model of sleep apnea by intermittent hypoxia.

Sleep apnea (SA) causes long-lasting changes in neuronal circuitry, which persist even in patients successfully treated for the acute effects of the disease. Evidence obtained from the intermittent hypoxia (IH) experimental model of SA has shown neuronal death, impairment in learning and memory and reactive gliosis that may account for cognitive and structural alterations observed in human patients. However, little is known about the mechanism controlling these deleterious effects that may be useful as therapeutic targets in SA. The Receptor for Advanced Glycation End products (RAGE) and its downstream effector Nuclear Factor Kappa B (NF-κB) have been related to neuronal death and astroglial conversion to the pro-inflammatory neurodegenerative phenotype. RAGE expression and its ligand S100B were shown to be increased in experimental models of SA. We here used dissociated mixed hippocampal cell cultures and male Wistar rats exposed to IH cycles and observed that NF-κB is activated in glial cells and neurons after IH. To disclose the relative contribution of the S100B/RAGE/NF-κB pathway to neuronal damage and reactive gliosis after IH we performed sequential loss of function studies using RAGE or S100B neutralizing antibodies, a herpes simplex virus (HSV)-derived amplicon vector that induces the expression of RAGEΔcyto (dominant negative RAGE) and a chemical blocker of NF-κB. Our results show that NF-κB activation peaks 3 days after IH exposure, and that RAGE or NF-κB blockage during this critical period significantly improves neuronal survival and reduces reactive gliosis. Both in vitro and in vivo, S100B blockage altered reactive gliosis but did not have significant effects on neuronal survival. We conclude that both RAGE and downstream NF-κB signaling are centrally involved in the neuronal alterations found in SA models, and that blockage of these pathways is a tempting strategy for preventing neuronal degeneration and reactive gliosis in SA.

Hippocampal NMDA receptors and the previous experience effect on memory.

N-methyl-D-aspartate receptors (NMDAR) are thought to be responsible for switching synaptic activity specific patterns into long-term changes in synaptic function and structure, which would support learning and memory. Hippocampal NMDAR blockade impairs memory consolidation in rodents, while NMDAR stimulation improves it. Adult rats that explored twice an open field (OF) before a weak though overthreshold training in inhibitory avoidance (IA), expressed IA long-term memory in spite of the hippocampal administration of MK-801, which currently leads to amnesia. Those processes would involve different NMDARs. The selective blockade of hippocampal GluN2B-containing NMDAR with ifenprodil after training promoted memory in an IA task when the training was weak, suggesting that this receptor negatively modulates consolidation. In vivo, after 1h of an OF exposure-with habituation to the environment-, there was an increase in GluN1 and GluN2A subunits in the rat hippocampus, without significant changes in GluN2B. Coincidentally, in vitro, in both rat hippocampal slices and neuron cultures there was an increase in GluN2A-NMDARs surface expression at 30min; an increase in GluN1 and GluN2A levels at about 1h after LTP induction was also shown. We hypothesize that those changes in NMDAR composition could be involved in the "anti-amnesic effect" of the previous OF. Along certain time interval, an increase in GluN1 and GluN2A would lead to an increase in synaptic NMDARs, facilitating synaptic plasticity and memory; while then, an increase in GluN2A/GluN2B ratio could protect the synapse and the already established plasticity, perhaps saving the specific trace.

NMDA receptor subunits in the adult rat hippocampus undergo similar changes after 5 minutes in an open field and after LTP induction.

NMDA receptor subunits change during development and their synaptic expression is modified rapidly after synaptic plasticity induction in hippocampal slices. However, there is scarce information on subunits expression after synaptic plasticity induction or memory acquisition, particularly in adults. GluN1, GluN2A and GluN2B NMDA receptor subunits were assessed by western blot in 1) adult rats that had explored an open field (OF) for 5 minutes, a time sufficient to induce habituation, 2) mature rat hippocampal neuron cultures depolarized by KCl and 3) hippocampal slices from adult rats where long term potentiation (LTP) was induced by theta-burst stimulation (TBS). GluN1 and GluN2A, though not GluN2B, were significantly higher 70 minutes--but not 30 minutes--after a 5 minutes session in an OF. GluN1 and GluN2A total immunofluorescence and puncta in neurites increased in cultures, as evaluated 70 minutes after KCl stimulation. Similar changes were found in hippocampal slices 70 minutes after LTP induction. To start to explore underlying mechanisms, hippocampal slices were treated either with cycloheximide (a translation inhibitor) or actinomycin D (a transcription inhibitor) during electrophysiological assays. It was corroborated that translation was necessary for LTP induction and expression. The rise in GluN1 depends on transcription and translation, while the increase in GluN2A appears to mainly depend on translation, though a contribution of some remaining transcriptional activity during actinomycin D treatment could not be rouled out. LTP effective induction was required for the subunits to increase. Although in the three models same subunits suffered modifications in the same direction, within an apparently similar temporal course, further investigation is required to reveal if they are related processes and to find out whether they are causally related with synaptic plasticity, learning and memory.

A novel dihydro-pyrazolo(3,4d)(1,2,4)triazolo(1,5a)pyrimidin-4-one (AJ23) is an antagonist at adenosine A(1) receptors and enhances consolidation of step-down avoidance.

Adenosine A(1) receptor antagonists are of potential value in the treatment of cognitive dysfunction. We have developed compound AJ23 (7-methyl-1-phenyl-1,8-dihydro-pyrazolo-(3,4d)(1,2,4)-triazolo(1,5a)-pyrimidin-4-one) as a novel, non-xanthine based antagonist at A(1) receptors. It has micromolar affinity at human A(1) receptors with a 45-fold selectivity for A(1) over A(2A) receptors and little affinity for many other receptors and transporters tested in a screening panel. AJ23 blocks A(1) receptors in the rat hippocampus, increasing the baseline size of excitatory post-synaptic potentials and blocking the inhibitory effects of adenosine. When administered directly into the rodent hippocampus this compound improves consolidation in a step-down avoidance learning task. The results suggest that AJ23 or derivatives may represent possible leads for further chemical development towards a chemically novel group of antagonists at A(1) receptors with potential value as cognitive enhancers.

Herpes simplex virus type 1-based amplicon vectors for fundamental research in neurosciences and gene therapy of neurological diseases.

Somatic manipulation of the nervous system without the involvement of the germinal line appears as a powerful counterpart of the transgenic strategy. The use of viral vectors to produce specific, transient and localized knockout, knockdown, ectopic expression or overexpression of a gene, leads to the possibility of analyzing both in vitro and in vivo molecular basis of neural function. In this approach, viral particles engineered to carry transgenic sequences are delivered into discrete brain regions, to transduce cells that will express the transgenic products. Amplicons are replication-incompetent helper-dependent vectors derived from herpes simplex virus type 1 (HSV-1), with several advantages that potentiate their use in neurosciences: (1) minimal toxicity: amplicons do not encode any virus proteins, are neither toxic for the infected cells nor pathogenic for the inoculated animals and elicit low levels of adaptive immune responses; (2) extensive transgene capacity to carry up to 150-kb of foreign DNA; i.e., entire genes with regulatory sequences could be delivered; (3) widespread cellular tropism: amplicons can experimentally infect several cell types including glial cells, though naturally the virus infects mainly neurons and epithelial cells; (4) since the viral genome does not integrate into cellular chromosomes there is low probability to induce insertional mutagenesis. Recent investigations on gene transfer into the brain using these vectors, have focused on gene therapy of inherited genetic diseases affecting the nervous system, such as ataxias, or on neurodegenerative disorders using experimental models of Parkinson's or Alzheimer's disease. Another group of studies used amplicons to investigate complex neural functions such as neuroplasticity, anxiety, learning and memory. In this short review, we summarize recent data supporting the potential of HSV-1 based amplicon vector model for gene delivery and modulation of gene expression in primary cultures of neuronal cells and into the brain of living animals.

Memory impairment in rats by hippocampal administration of the serine protease subtilisin.

Since the serine protease subtilisin has been reported to generate a novel form of long-term depression (LTD) in rat hippocampal slices, the present work was designed to determine whether it has any effect on learning and memory processes. Rats were used to examine the effects of subtilisin, injected directly into the dorsal hippocampus, on task performance in a step-through inhibitory avoidance of a mild footshock. The administration of 100 ng of subtilisin into each hippocampus, immediately after training, was sufficient to induce a detectable learning deficit with a footshock stimulus of 0.5 mA. Higher doses produced dose-related impairments in memory consolidation. These effects were not the result of irreversible toxicity, since rats trained with a higher amplitude footshock (0.75 mA) were able to perform as control animals; therefore, the amnesic effect was not further evident. Furthermore, the administration of subtilisin before avoidance training did not produce any detectable effect on performance during the training or test sessions, indicating that neither acquisition nor consolidation was affected. It is concluded that the post-training administration of a serine protease inhibitor is able to produce robust deficits of memory consolidation consistent with its ability to generate LTD, raising the possibility that related molecules could play physiological or pathological roles in the modulation of learning and memory.

N-methyl-D-aspartate receptors are required for synaptic targeting of Alzheimer's toxic amyloid-ß peptide oligomers.

Soluble amyloid-ß peptide (Aß) oligomers, known to accumulate in Alzheimer's disease brains, target excitatory post-synaptic terminals. This is thought to trigger synapse deterioration, a mechanism possibly underlying memory loss in early stage Alzheimer's disease. A major unknown is the identity of the receptor(s) targeted by oligomers at synapses. Because oligomers have been shown to interfere with N-methyl-d-aspartate receptor (NMDAR) function and trafficking, we hypothesized that NMDARs might be required for oligomer binding to synapses. An amplicon vector was used to knock-down NMDARs in mature hippocampal neurons in culture, yielding 90% reduction in dendritic NMDAR expression and blocking neuronal oxidative stress induced by Aß oligomers, a pathological response that has been shown to be mediated by NMDARs. Remarkably, NMDAR knock-down abolished oligomer binding to dendrites, indicating that NMDARs are required for synaptic targeting of oligomers. Nevertheless, oligomers do not appear to bind directly to NMDARs as indicated by the fact that both oligomer-attacked and non-attacked neurons exhibit similar surface levels of NMDARs. Furthermore, pre-treatment of neurons with insulin down-regulates oligomer-binding sites in the absence of a parallel reduction in surface levels of NMDARs. Establishing that NMDARs are key components of the synaptic oligomer binding complex may illuminate the development of novel approaches to prevent synapse failure triggered by Aß oligomers.

Muscarinic inhibition of hippocampal and striatal adenylyl cyclase is mainly due to the M(4) receptor.

The five muscarinic acetylcholine receptors (M(1)-M(5)) are differentially expressed in the brain. M(2) and M(4) are coupled to inhibition of stimulated adenylyl cyclase, while M(1), M(3) and M(5) are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2-affinity order M(1) > or = M(4) >> others-and MT3-highly selective M(4) antagonist-did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M(2), M(4) would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M(4) receptor would be responsible for this regulation.

M4 muscarinic receptors are involved in modulation of neurotransmission at synapses of Schaffer collaterals on CA1 hippocampal neurons in rats.

All five subtypes of muscarinic acetylcholine receptors (mAChR; M(1)-M(5)) are expressed in the hippocampus, where they are involved both in cognitive functions and in synaptic plasticity, such as long-term potentiation (LTP). Muscarinic toxins (MTs) are small proteins from mamba snake venoms that display exquisite discrimination between mAChRs. MT1 acts as an agonist at M(1) and an antagonist at M(4) receptors, with similar affinities for both. MT3, the most selective antagonist available for M(4) receptors, infused into the CA1 region immediately after training caused amnesia in the rat, indicating the participation of M(4) receptors in memory consolidation. Our goal was to investigate the participation of M(4) receptor in neurotransmission at the hippocampal Schaffer collaterals-CA1 synapses. Two different preparations were used: 1) field potential recordings in freshly prepared rat hippocampal slices with high-frequency stimulation to induce potentiation and 2) whole-cell voltage clamp in cultured hippocampal organotypic slices with paired stimuli. In preparation 1, a dose of MT3 that was previously shown to cause amnesia blocked LTP; the nonselective antagonist scopolamine blocked LTP without affecting basal transmission, although it was depressed with higher concentration. In preparation 2, basal transmission was decreased and LTP induction was prevented by an MT3 concentration that would bind mainly to M(4) receptors. Although M(1) receptors appeared to modulate transmission positively at these excitatory synapses, M(1) activation concomitant with M(4) blockade (by MT1) only allowed a brief, short-term potentiation. Accordingly, M(4) blockade by MT3 strongly supports a permissive role of M(4) receptors and suggests their necessary participation in synaptic plasticity at these synapses.

Amplicon vectors as outstanding tools to study and modify cognitive functions.

This review summarizes recent data on the use of HSV-1-based amplicon vectors for in vivo gene delivery to the brains of rats and mice to study and modify behaviour. Here we describe studies that have focused on cognitive functions like learning and memory. In addition, the use of amplicons in other behavioural studies, like addiction, social interaction, anxiety and stress, will be briefly updated. Several remarkable findings have been achieved, thanks to the use of these very efficient and non-toxic naturally neurotropic vectors, most particularly the consistent observation that genetic manipulation of a rather limited number of neurons in restricted regions of the brain, could result in significant behavioural changes, a notion that is therefore emerging as a common unifying hypothesis, thanks to these works.