Rapid increase in clusters of presynaptic proteins at onset of long-lasting potentiation.

A change in the efficiency of synaptic communication between neurons is thought to underlie learning. Consistent with recent studies of such changes, we find that long-lasting potentiation of synaptic transmission between cultured hippocampal neurons is accompanied by an increase in the number of clusters of postsynaptic glutamate receptors containing the subunit GluR1. In addition, potentiation is accompanied by a rapid and long-lasting increase in the number of clusters of the presynaptic protein synaptophysin and the number of sites at which synaptophysin and GluR1 are colocalized. These results suggest that potentiation involves rapid coordinate changes in the distribution of proteins in the presynaptic neuron as well as the postsynaptic neuron.

Correction: Abolishing Tau cleavage by caspases at Aspartate421 causes memory/synaptic plasticity deficits and pre-pathological Tau alterations.

Text for Correction.

Abolishing Tau cleavage by caspases at Aspartate421 causes memory/synaptic plasticity deficits and pre-pathological Tau alterations.

TAU mutations are genetically linked to fronto-temporal dementia (FTD) and hyper-phosphorylated aggregates of Tau form neurofibrillary tangles (NFTs) that constitute a pathological hallmark of Alzheimer disease (AD) and FTD. These observations indicate that Tau has a pivotal role in the pathogenesis of neurodegenerative disorders. Tau is cleaved by caspases at Aspartate421, to form a Tau metabolite known as δTau; δTau is increased in AD, due to the hyper-activation of caspases in AD brains. δTau is considered a critical toxic moiety underlying neurodegeneration, which initiates and facilitates NFT formation. As Tau is a therapeutic target in neurodegeneration, it is important to rigorously determine whether δTau is a toxic Tau species that should be pharmacologically attacked. To directly address these questions, we have generated a knock-in (KI) mouse called TauDN-that expresses a Tau mutant that cannot be cleaved by caspases. TauDN mice present short-term memory deficits and synaptic plasticity defects. Moreover, mice carrying two mutant Tau alleles show increased total insoluble hyper-phosphorylated Tau in the forebrain. These data are in contrast with the concept that δTau is a critical toxic moiety underlying neurodegeneration, and suggest that cleavage of Tau by caspases represents a negative feedback mechanism aimed to eliminate toxic Tau species. Alternatively, it is possible that either a reduction or an increase in δTau leads to synaptic dysfunction, memory impairments and Tau pathology. Both possibilities will have to be considered when targeting caspase cleavage of Tau in AD therapy.

Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory.

Non-fibrillar soluble oligomeric forms of amyloid-ß peptide (oAß) and tau proteins are likely to play a major role in Alzheimer's disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAß initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aß, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAß levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAß to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aß on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aß and tau pathology.

Presynaptic role of cGMP-dependent protein kinase during long-lasting potentiation.

Previous research has suggested that cGMP-dependent protein kinases (cGKs) may play a role in long-term potentiation in hippocampus, but their site of action has been unknown. We examined this question at synapses between pairs of hippocampal neurons in dissociated cell culture. Injection of a specific peptide inhibitor of cGK into the presynaptic but not the postsynaptic neuron blocked long-lasting potentiation induced by tetanic stimulation of the presynaptic neuron. As controls, injection of a scrambled peptide or a peptide inhibitor of cAMP-dependent protein kinase into either neuron did not block potentiation. Conversely, injection of the alpha isozyme of cGK type I into the presynaptic but not the postsynaptic neuron produced activity-dependent potentiation that did not require NMDA receptor activation. Evidence from Western blots, reverse transcription-PCR, activity assays, and immunocytochemistry indicates that endogenous cGK type I is present in the neurons, including presynaptic terminals. These results support the idea that cGK plays an important presynaptic role during the induction of long-lasting potentiation in hippocampal neurons.

The keystone of Alzheimer pathogenesis might be sought in Aß physiology.

For several years Amyloid-beta peptide (Aß) has been considered the main pathogenetic factor of Alzheimer's disease (AD). According to the so called Amyloid Cascade Hypothesis the increase of Aß triggers a series of events leading to synaptic dysfunction and memory loss as well as to the structural brain damage in the later stage of the disease. However, several evidences suggest that this hypothesis is not sufficient to explain AD pathogenesis, especially considering that most of the clinical trials aimed to decrease Aß levels have been unsuccessful. Moreover, Aß is physiologically produced in the healthy brain during neuronal activity and it is needed for synaptic plasticity and memory. Here we propose a model interpreting AD pathogenesis as an alteration of the negative feedback loop between Aß and its physiological receptors, focusing on alpha7 nicotinic acetylcholine receptors (α7-nAchRs). According to this vision, when Aß cannot exert its physiological function a negative feedback mechanism would induce a compensatory increase of its production leading to an abnormal accumulation that reduces α7-nAchR function, leading to synaptic dysfunction and memory loss. In this perspective, the indiscriminate Aß removal might worsen neuronal homeostasis, causing a further impoverishment of learning and memory. Even if further studies are needed to better understand and validate these mechanisms, we believe that to deepen the role of Aß in physiological conditions might represent the keystone to elucidate important aspects of AD pathogenesis.

The distribution of excitatory amino acid receptors on acutely dissociated dorsal horn neurons from postnatal rats.

Excitatory amino acid receptor distribution was mapped on acutely dissociated neurons from postnatal rat spinal cord dorsal horn. N-methyl D-aspartate, quisqualate and kainate were applied to multiple locations along the somal and dendritic surfaces of voltage-clamped neurons by means of a pressure application system. To partially compensate for the decrement of response amplitude due to current loss between the site of activation on the dendrite and the recording electrode at the soma, a solution containing 0.15 M KCl was applied on the cell bodies and dendrites of some cells to estimate an empirical length constant. In the majority of the cells tested, the dendritic membrane had regions of higher sensitivity to excitatory amino acid agonists than the somatic membrane, with dendritic response amplitudes reaching more than seven times those at the cell body. A comparison of the relative changes in sensitivity between each combination of two of the three excitatory amino acid agonists along the same dendrite showed different patterns of agonist sensitivity along the dendrite in the majority of the cells. These data were obtained from dorsal horn neurons that had developed and formed synaptic connections in vivo. They demonstrate that in contrast to observations made on ventral horn neurons, receptor density for all the excitatory amino acid receptors on dorsal horn neurons, including the N-methyl-D-aspartate receptor, are generally higher on the dendrites than on the soma. Further, these results are similar to those obtained from dorsal horn neurons grown in culture.

Nitric oxide as a retrograde messenger during long-term potentiation in hippocampus.

Nitric oxide (NO) is widespread in the nervous system and is thought to play a role in a variety of different neuronal functions, including learning and memory (see other chapters, this volume). A number of behavioral studies have indicated that NO is involved in several types of learning such as motor learning (Yanagihara and Kondo, 1996), avoidance learning (Barati and Kopf, 1996; Myslivecek et al., 1996), olfactory learning (Okere et. al., 1996; Kendrick et al., 1997), and spatial learning (Holscher et al., 1995; Yamada et al., 1996) (for review of earlier papers see Hawkins, 1996). Moreover, NO is thought to be involved in neuronal plasticity contributing to these different types of learning in different brain areas including the cerebellum (chapter by R. Tsien, this volume) and hippocampus. In this chapter we review evidence on the role of NO in long-term potentiation (LTP), a type of synaptic plasticity in hippocampus that is believed to contribute to declarative forms of learning such as spatial learning.

Morphological changes associated with long-term potentiation.

Long-term potentiation (LTP) is a long-lasting form of synaptic plasticity induced by brief repetitive afferent stimulation that is thought to be associated with learning and memory. It is most commonly studied in the hippocampus where it may last for several weeks, and involves the synthesis of new proteins that might play a structural role. In this review we summarize the evidence in favor of modifications of neuronal architecture during LTP. We focus our attention on changes occurring at the level of single synapses, including components of postsynaptic dendrites (dendritic spines, the postsynaptic density, and synaptic curvature), of presynaptic terminals, and the formation of new synapses. We conclude that although many morphological changes at various sites have been observed during LTP, there is no definitive proof in favor of structural changes associated with LTP. However, morphological modifications remain a valid candidate for mechanisms of learning and memory.

Alpha-synuclein: between synaptic function and dysfunction.

Alpha-synuclein belongs to a family of vertebrate proteins, encoded by three different genes: alpha, ss, and gamma. The protein has become of interest to the neuroscience community in the last few years after the discovery that a mutation in the alpha-synuclein gene is associated with familial autosomal-dominant early-onset forms of Parkinson Disease. However, it is not yet clear how the protein is involved in the disease. Several studies have suggested that alpha-synuclein plays a role in neurotransmitter release and synaptic plasticity. This hypothesis might help elucidate how alpha-synuclein malfunctioning contributes to the development of a series of disorders known as synucleinopathies.

Glutamate receptor agonist-induced inward currents in spinal dorsal horn neurons dissociated from the adult rats.

Inward currents to glutamate receptor agonists, quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA) were examined in spinal dorsal horn neurons by whole-cell voltage-clamp techniques after acute dissociation. Neurons were dissociated from the superficial dorsal horn (laminae I/II) of the adult rat (8-16 weeks old) spinal cords by enzymatic and mechanical treatment. The KA-induced current was sustained during KA application, while the QA- and NMDA-induced currents were attenuated. The NMDA response was augmented dose-dependently by addition of glycine (10(-7)-5 X 10(-6) M) and became obscure in the absence of glycine. The NMDA current was depressed by D-2-amino-5-phosphonovaleric acid (APV). Analyses of dose-response curves of these inward currents indicate that both the QA and KA currents were competitively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), while the NMDA current was blocked non-competitively.

Nitric oxide acts as a retrograde messenger during long-term potentiation in cultured hippocampal neurons.

We examined long-term potentiation (LTP) at synapses between hippocampal neurons in dissociated cell culture following presynaptic, postsynaptic, or extracellular application of a nitric oxide (NO) scavenger, an inhibitor of NO synthase, and a membrane-impermeant NO donor that releases NO only upon photolysis with UV light. Our results indicate that NO is produced in the postsynaptic neuron, travels through the extracellular space, and acts directly in the presynaptic neuron to produce long-term potentiation, supporting the hypothesis that NO acts as a retrograde messenger during LTP.

Nerve stump effects in muscle are independent of synaptic connections and are temporally correlated with nerve degeneration phenomena.

Close or distant denervation of the rat soleus muscle indicated that (1) longer soleus nerve stumps delay the onset of axon terminal degeneration and of muscle membrane changes (spike resistance to TTX) by strictly comparable times, and (2) the stump-induced delay of the muscle effect is independent of synaptic connections, because it is also obtained (RMP fall and TTX-resistance development) when sectioning a foreign nerve previously transplanted on the soleus surface but not making synaptic contacts. Both lines of evidence are consistent with the interpretation that, as far as the extrajunctional membrane properties are concerned, the effect of the length of the nerve stump on muscle is mediated by nerve terminal breakdown.

Effects of reinnervation with normal and tetrodotoxin-inactive nerves on resting membrane potential of rat skeletal muscle.

Resting membrane potentials (RMPs) have been recorded in vitro near the end-plate region of rat soleus muscles reinnervated with tetrodotoxin-inactive nerves and compared with those of denervated muscles whose reinnervation had been prevented. The two muscle groups exhibited the same low values of RMP typical of denervated muscles. In control muscles of rats in which impulse conduction was left unimpaired, reinnervation induced the expected increase in RMP values towards normal. It is suggested that, at least for this property, reinnervation restores to normal the muscle fibre membrane essentially through the return of activity.

Differential distribution of excitatory amino acid receptors on embryonic rat spinal cord neurons in culture.

1. Excitatory amino acid (EAA) receptors mediate synaptic transmission in many areas of the vertebrate CNS. To map the distribution of the EAA receptors, three agonists selective for EAA receptor subtypes [kainate, quisqualate, and N-methyl-D-aspartate (NMDA)] were applied by pressure to the cell bodies and neurites of voltage-clamped, embryonic rat spinal cord neurons in culture. 2. Current loss along the neurite between the site of activation and the recording electrode at the soma was estimated independently of variations in receptor density by focal application of high [K+] solution. This estimate was used to compensate agonist-evoked responses for current loss due to leak. K(+)-evoked current amplitudes progressively decreased as applications were made at more distal positions along the neurite. 3. Response amplitudes to EAAs showed a monotonic decay, similar to that with high [K+] solution, in only a few of the cells tested with each agonist. The majority of neurons had areas of high agonist sensitivity along the neurites, implying a nonuniform density of receptors. Such regions sometimes occurred at the most proximal segment of the neurite. Most neurites had distal regions of agonist sensitivity that ranged from 40 to 300% higher than at the soma. 4. The relative distributions of the three EAA receptors were compared by applying two agonists to the same sites along neurites and observing variations in normalized response amplitudes. When comparing NMDA versus non-NMDA receptor distributions on the same neurite, approximately 40% of the cells had similar patterns to two agonists, and the remaining 60% had different patterns. When sensitivity to the two non-NMDA agonists kainate and quisqualate were compared, about one-half of the cells tested had different patterns of agonist sensitivity. 5. Areas of high sensitivity persisted after block of calcium channels by addition of La3+ to the bath solution and after prevention of evoked transmitter release by a low [Ca2+]/high [Mg2+] solution. 6. These results show that spinal cord neurons can have regions of high agonist sensitivity to NMDA, kainate, and quisqualate along their neurites and that the sensitivity to any one of the EAA receptor subtypes can be elevated independently of the others.

Nitric oxide and carbon monoxide as possible retrograde messengers in hippocampal long-term potentiation.

We have been investigating the hypothesis that the membrane-permeant molecules nitric oxide (NO) and carbon monoxide (CO) may act as retrograde messengers during long-term potentiation (LTP). Inhibitors of either NO synthase or heme oxygenase, the enzyme that produces CO, blocked induction of LTP in the CA1 region of hippocampal slices. Brief application of either NO or CO to slices produced a rapid and long-lasting increase in the size of synaptic potentials if, and only if, the application occurred at the same time as weak tetanic stimulation of the presynaptic fibers. The long-term enhancement by NO or CO was spatially restricted to synapses from active presynaptic fibers and appeared to involve mechanisms utilized by LTP, occluding the subsequent induction of LTP by strong tetanic stimulation. The enhancement by NO or CO was not blocked by the NMDA receptor blocker APV, suggesting that NO and CO act downstream from the NMDA receptor. In other systems, both NO and CO produce many of their effects by activation of soluble guanylyl cyclase and cGMP-dependent protein kinase. An inhibitor of soluble guanylyl cyclase blocked the induction of normal LTP. Conversely, the membrane-permeable analog 8-Br-cGMP produced a rapid onset and long-lasting synaptic enhancement if, and only if, it was applied at the same time as weak presynaptic stimulation. Similarly, two inhibitors of cGMP-dependent protein kinase blocked the induction of normal LTP, and a selective activator of cGMP-dependent protein kinase produced activity-dependent long-lasting synaptic enhancement. 8-Br-cGMP also produced an activity-dependent, long-lasting increase in the amplitude of evoked synaptic currents between pairs of hippocampal neurons in dissociated cell culture. In addition, 8-Br-cGMP, like NO, produced a long-lasting increase in the frequency of spontaneous miniature synaptic currents. These results are consistent with the hypothesis that NO and CO, either alone or in combination, serve as retrograde messengers that produce activity-dependent presynaptic enhancement, perhaps by stimulating soluble guanylyl cyclase and cGMP-dependent protein kinase, during LTP in hippocampus.

Activity-dependent long-term enhancement of transmitter release by presynaptic 3',5'-cyclic GMP in cultured hippocampal neurons.

Long-term potentiation (LTP) in hippocampus is a type of synaptic plasticity that is thought to be involved in learning and memory. Several lines of evidence suggest that LTP involves 3',5'-cyclic GMP (cGMP), perhaps as an activity-dependent presynaptic effector of one or more retrograde messengers (refs 2-12, but see ref. 13). However, previous results are also consistent with postsynaptic effects of cGMP. This is difficult to test in hippocampal slices, but more rigorous tests are possible in dissociated cell culture. We have therefore developed a reliable method for producing N-methyl-D-aspartate (NMDA) receptor-dependent LTP at synapses between individual hippocampal pyramidal neurons in culture. We report that inhibitors of guanylyl cyclase or of cGMP-dependent protein kinase block potentiation by either tetanic stimulation or low-frequency stimulation paired with postsynaptic depolarization. Conversely, application of 8-Br-cGMP to the bath or injection of cGMP into the presynaptic neuron produces activity-dependent long-lasting potentiation. The potentiation by cGMP involves an increase in transmitter release that is in part independent of changes in the presynaptic action potential. These results support a presynaptic role for cGMP in LTP.

Fibrillatory activity and other membrane changes in partially denervated muscles.

Rat soleus muscles were partially or totally denervated by sectioning the radicular nerve L5 or the radicular nerves L3 through L6, respectively. Three days after these procedures, fibrillation potentials were not observed in the case of partial denervation, whereas they were clearly detectable after total denervation. At later times, spontaneous spike activity also developed in the partially denervated muscles. The difference in time of onset of fibrillation between partially and totally denervated muscles was confirmed by a more gradual increase in the number of acetylcholine receptors and a greater sensitivity to tetrodotoxin of the former muscles. These differences between partially and totally denervated muscles are interpreted on the basis of the different amounts of nerve breakdown products generated in the two situations.

[Role of NGF in the development of primary sensory neurons in the rat]. / Ruolo dell 'NGF nello sviluppo dei neuroni sensitivi primari nel ratto.

Two populations of neurons are present in the dorsal root ganglia: i) the first which is connected with the neuromuscular spindles is localized in the mediodorsal (MD) part of the ganglion; ii) the second which is connected with the cutaneous receptors is localized in the ventrolateral (VL) part of the ganglion. The growth of MD but not of VL neurons in vitro is dependent upon the presence of the NGF. In order to study the presence in vivo of such differential effect of the NGF on the two neuronal populations, we have injected rat embryos with NGF antiserum (AS-NGF) and recorded the compound action potentials of different hindlimb nerves. We found that the sensory component, in the studied muscular nerves of the hindlimb, is seriously depressed in treated animals. These results indicate that the animals treated with AS-NGF suffer of a depression of activity in the afferents from the muscle spindles. This is probably due to a decrease in the number of these fibers in their muscle nerves.