Wnt-5a occludes Aβ oligomer-induced depression of glutamatergic transmission in hippocampal neurons

BackgroundSoluble amyloid-β (Aβ;) oligomers have been recognized to be early and key intermediates in Alzheimer's disease (AD)-related synaptic dysfunction. Aβ oligomers block hippocampal long-term potentiation (LTP) and impair rodent spatial memory. Wnt signaling plays an important role in neural development, including synaptic differentiation.ResultsWe report here that the Wnt signaling activation prevents the synaptic damage triggered by Aβ oligomers. Electrophysiological analysis of Schaffer collaterals-CA1 glutamatergic synaptic transmission in hippocampal slices indicates that Wnt-5a increases the amplitude of field excitatory postsynaptic potentials (fEPSP) and both AMPA and NMDA components of the excitatory postsynaptic currents (EPSCs), without modifying the paired pulse facilitation (PPF). Conversely, in the presence of Aβ oligomers the fEPSP and EPSCs amplitude decreased without modification of the PPF, while the postsynaptic scaffold protein (PSD-95) decreased as well. Co-perfusion of hippocampal slices with Wnt-5a and Aβ oligomers occludes against the synaptic depression of EPSCs as well as the reduction of PSD-95 clusters induced by Aβ oligomers in neuronal cultures. Taken together these results indicate that Wnt-5a and Aβ oligomers inversely modulate postsynaptic components.ConclusionThese results indicate that post-synaptic damage induced by Aβ oligomers in hippocampal neurons is prevented by non-canonical Wnt pathway activation.

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abstract: At vertebrate neuromuscular junctions (NMJs), Agrin plays pivotal roles in synapse development, but molecules that activate synapse formation at central synapses are largely unknown. Members of the Wnt family are well established as morphogens, yet recently they have also been implicated in synapse maturation. Here we demonstrate that the Drosophila Wnt, Wingless (Wg), is essential for synapse development. We show that Wg and its receptor are expressed at glutamatergic NMJs, and that Wg is secreted by synaptic boutons. Loss of Wg leads to dramatic reductions in target-dependent synapse formation, and new boutons either fail to develop active zones and postsynaptic specializations or these are strikingly aberrant. We suggest that Wg signals the coordinated development of pre- and postsynaptic compartments.

title: Natural Oligomers of the Alzheimer Amyloid-β Protein Induce Reversible Synapse Loss by Modulating an NMDA-Type Glutamate Receptor-Dependent Signaling Pathway
abstract: Alzheimer's disease (AD) is characterized by decreased synapse density in hippocampus and neocortex, and synapse loss is the strongest anatomical correlate of the degree of clinical impairment. Although considerable evidence supports a causal role for the amyloid-β protein (Aβ) in AD, a direct link between a specific form of Aβ and synapse loss has not been established. We demonstrate that physiological concentrations of naturally secreted Aβ dimers and trimers, but not monomers, induce progressive loss of hippocampal synapses. Pyramidal neurons in rat organotypic slices had markedly decreased density of dendritic spines and numbers of electrophysiologically active synapses after exposure to picomolar levels of soluble oligomers. Spine loss was reversible and was prevented by Aβ-specific antibodies or a small-molecule modulator of Aβ aggregation. Mechanistically, Aβ-mediated spine loss required activity of NMDA-type glutamate receptors (NMDARs) and occurred through a pathway involving cofilin and calcineurin. Furthermore, NMDAR-mediated calcium influx into active spines was reduced by Aβ oligomers. Partial blockade of NMDARs by pharmacological antagonists was sufficient to trigger spine loss. We conclude that soluble, low-n oligomers of human Aβ trigger synapse loss that can be reversed by therapeutic agents. Our approach provides a quantitative cellular model for elucidating the molecular basis of Aβ-induced neuronal dysfunction.

title: Amyloid-β protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory
abstract: Alzheimer's disease constitutes a rising threat to public health. Despite extensive research in cellular and animal models, identifying the pathogenic agent present in the human brain and showing that it confers key features of Alzheimer's disease has not been achieved. We extracted soluble amyloid-β protein (Aβ) oligomers directly from the cerebral cortex of subjects with Alzheimer's disease. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD) and reduced dendritic spine density in normal rodent hippocampus. Soluble Aβ from Alzheimer's disease brain also disrupted the memory of a learned behavior in normal rats. These various effects were specifically attributable to Aβ dimers. Mechanistically, metabotropic glutamate receptors were required for the LTD enhancement, and N-methyl D-aspartate receptors were required for the spine loss. Co-administering antibodies to the Aβ N-terminus prevented the LTP and LTD deficits, whereas antibodies to the midregion or C-terminus were less effective. Insoluble amyloid plaque cores from Alzheimer's disease cortex did not impair LTP unless they were first solubilized to release Aβ dimers, suggesting that plaque cores are largely inactive but sequester Aβ dimers that are synaptotoxic. We conclude that soluble Aβ oligomers extracted from Alzheimer's disease brains potently impair synapse structure and function and that dimers are the smallest synaptotoxic species.

title: High-Level Neuronal Expression of Aβ1–42 in Wild-Type Human Amyloid Protein Precursor Transgenic Mice: Synaptotoxicity without Plaque Formation
abstract: Amyloid plaques are a neuropathological hallmark of Alzheimer's disease (AD), but their relationship to neurodegeneration and dementia remains controversial. In contrast, there is a good correlation in AD between cognitive decline and loss of synaptophysin-immunoreactive (SYN-IR) presynaptic terminals in specific brain regions. We used expression-matched transgenic mouse lines to compare the effects of different human amyloid protein precursors (hAPP) and their products on plaque formation and SYN-IR presynaptic terminals. Four distinct minigenes were generated encoding wild-type hAPP or hAPP carrying mutations that alter the production of amyloidogenic Aβ peptides. The platelet-derived growth factor β chain promoter was used to express these constructs in neurons. hAPP mutations associated with familial AD (FAD) increased cerebral Aβ1–42 levels, whereas an experimental mutation of the β-secretase cleavage site (671M→I) eliminated production of human Aβ. High levels of Aβ1–42 resulted in age-dependent formation of amyloid plaques in FAD-mutant hAPP mice but not in expression-matched wild-type hAPP mice. Yet, significant decreases in the density of SYN-IR presynaptic terminals were found in both groups of mice. Across mice from different transgenic lines, the density of SYN-IR presynaptic terminals correlated inversely with Aβ levels but not with hAPP levels or plaque load. We conclude that Aβ is synaptotoxic even in the absence of plaques and that high levels of Aβ1–42are insufficient to induce plaque formation in mice expressing wild-type hAPP. Our results support the emerging view that plaque-independent Aβ toxicity plays an important role in the development of synaptic deficits in AD and related conditions.

title: Altered expression of synaptic proteins occurs early during progression of Alzheimer’s disease
abstract: The expression levels of three synaptic proteins (synaptophysin, synaptotagmin, and growth-associated protein 43 [GAP43]) in AD cases clinically classified by Clinical Dementia Rating (CDR) score were analyzed. Compared with control subjects (CDR = 0), mild (early) AD (CDR = 0.5 to 1) cases had a 25% loss of synaptophysin immunoreactivity. Levels of synaptotagmin and GAP43 were unchanged in mild AD, but cases with CDR of >1 had a progressive decrement in these synaptic proteins. Thus, synaptic injury in frontal cortex is an early event in AD.

title: Structure-function implications in Alzheimer's disease: effect of Abeta oligomers at central synapses.
abstract: Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the growing population of elderly people. A characteristic of AD is the accumulation of plaques in the brain of AD patients, and theses plaques mainly consist of aggregates of amyloid beta-peptide (Abeta). All converging lines of evidence suggest that progressive accumulation of the Abeta plays a central role in the genesis of Alzheimer's disease and it was long understood that Abeta had to be assembled into extracellular amyloid fibrils to exert its cytotoxic effects. This process could be modulated by molecular chaperones which inhibit or accelerate the amyloid formation. The enzyme Acetylcholinesterase (AChE) induces Abeta fibrils formation, forming a stable complex highly neurotoxic. On the other hand, laminin inhibit the Abeta fibrils formation and depolymerizate Abeta fibrils also. Over the past decade, data have emerged from the use of several sources of Abeta (synthetic, cell culture, transgenic mice and human brain) to suggest that intermediate species called Abeta oligomers are also injurious. Accumulating evidence suggests that soluble forms of Abeta are indeed the proximate effectors of synapse loss and neuronal injury. On the other hand, the member of the Wnt signaling pathway, beta-catenin was markedly reduced in AD patients carrying autosomal dominant PS-1. Also, neurons incubated with Abeta revealed a significant dose-dependent decrease in the levels of cytosolic beta-catenin an effect which was reversed in cells co-incubated with increasing concentrations of lithium, an activator of Wnt signaling pathway. Wnt signaling blocks the behavioural impairments induced by hippocampal injection of Abeta, therefore the activation of Wnt signaling protects against the Abeta neurotoxicity. Here we review recent progress about Abeta structure and function, from the formation of amyloid fibrils and some molecular chaperones which modulate the amyloidogenesic process to synaptic damage induce by Abeta oligomers.

title: Cellular Prion Protein Mediates Impairment of Synaptic Plasticity by Amyloid-β Oligomers
abstract: A pathological hallmark of Alzheimer’s disease is an accumulation of insoluble plaque containing the amyloid-β peptide of 40–42 amino acid residues. Prefibrillar, soluble oligomers of amyloid-β have been recognized to be early and key intermediates in Alzheimer’s-disease-related synaptic dysfunction. At nanomolar concentrations, soluble amyloid-β oligomers block hippocampal long-term potentiation, cause dendritic spine retraction from pyramidal cells and impair rodent spatial memory. Soluble amyloid-β oligomers have been prepared from chemical syntheses, transfected cell culture supernatants, transgenic mouse brain and human Alzheimer’s disease brain. Together, these data imply a high-affinity cell-surface receptor for soluble amyloid-β oligomers on neurons—one that is central to the pathophysiological process in Alzheimer’s disease. Here we identify the cellular prion protein (PrPC) as an amyloid-β-oligomer receptor by expression cloning. Amyloid-β oligomers bind with nanomolar affinity to PrPC, but the interaction does not require the infectious PrPSc conformation. Synaptic responsiveness in hippocampal slices from young adult PrP null mice is normal, but the amyloid-β oligomer blockade of long-term potentiation is absent. Anti-PrP antibodies prevent amyloid-β-oligomer binding to PrPC and rescue synaptic plasticity in hippocampal slices from oligomeric amyloid-β. Thus, PrPC is a mediator of amyloid-β-oligomer-induced synaptic dysfunction, and PrPC-specific pharmaceuticals may have therapeutic potential for Alzheimer’s disease.

title: Wnt-5a/JNK Signaling Promotes the Clustering of PSD-95 in Hippocampal Neurons*
abstract: During the formation of synapses, specific regions of pre- and postsynaptic cells associate to form a single functional transmission unit. In this process, synaptogenic factors are necessary to modulate pre- and postsynaptic differentiation. In mammals, different Wnt ligands operate through canonical and non-canonical Wnt pathways, and their precise functions to coordinate synapse structure and function in the mature central nervous system are still largely unknown. Here, we studied the effect of different Wnt ligands on postsynaptic organization. We found that Wnt-5a induces short term changes in the clustering of PSD-95, without affecting its total levels. Wnt-5a promotes the recruitment of PSD-95 from a diffuse dendritic cytoplasmic pool to form new PSD-95 clusters in dendritic spines. Moreover, Wnt-5a acting as a non-canonical ligand regulates PSD-95 distribution through a JNK-dependent signaling pathway, as demonstrated by using the TAT-TI-JIP peptide in mature hippocampal neurons. Finally, using adult rat hippocampal slices, we found that Wnt-5a modulates glutamatergic synaptic transmission through a postsynaptic mechanism. Our studies indicate that the Wnt-5a/JNK pathway modulates the postsynaptic region of mammalian synapse directing the clustering and distribution of the physiologically relevant scaffold protein, PSD-95.

title: Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development
abstract: Tissue and molecular heterogeneities are present in the developing secondary palate along the anteroposterior (AP) axis in mice. Here, we show that Wnt5a and its receptor Ror2 are expressed in a graded manner along the AP axis of the palate. Wnt5a deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histological, cellular and molecular levels in the anterior and posterior regions of the palate. We demonstrate that there is directional cell migration within the developing palate. In the absence of Wnt5a, this directional cell migration does not occur. Genetic studies and in vitro organ culture assays further demonstrate a role for Ror2 in mediating Wnt5a signaling in the regulation of cell proliferation and migration during palate development. Our results reveal distinct regulatory roles for Wnt5a in gene expression and cell proliferation along the AP axis of the developing palate, and an essential role for Wnt5a in the regulation of directional cell migration.

title: PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating
abstract: Neuronal NMDA receptors (NMDARs) colocalize with postsynaptic density protein-95 (PSD-95), a putative NMDAR anchoring protein and core component of the PSD, at excitatory synapses. PKC activation and PSD-95 expression each enhance NMDAR channel opening rate and number of functional channels at the cell surface. Here we show in Xenopus oocytes that PSD-95 and PKC potentiate NMDA gating and trafficking in a nonadditive manner. PSD-95 and PKC each enhance NMDA channel activity, with no change in single-channel conductance, reversal potential or mean open time. PSD-95 and PKC each potentiate NMDA channel opening rate (kβ) and number of functional channels at the cell surface (N), as indicated by more rapid current decay and enhanced charge transfer in the presence of the open channel blocker MK-801. PSD-95 and PKC each increase NMDAR surface expression, as indicated by immunofluorescence. PKC potentiates NMDA channel function and NMDAR surface expression to the same final absolute values in the absence or presence of PSD-95. Thus, PSD-95 partially occludes PKC potentiation. We further show that Ser-1462, a putative phosphorylation target within the PDZ-binding motif of the NR2A subunit, is required for PSD-95-induced potentiation and partial occlusion of PKC potentiation. Coimmunoprecipitation experiments with cortical neurons in culture indicate that PKC activation promotes assembly of NR2 with NR1, and that the newly assembled NMDARs are not associated with PSD-95. These findings predict that synaptic scaffolding proteins and protein kinases convergently modulate NMDAR gating and trafficking at synaptic sites.

title: Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development
abstract: Dendritic arborization is required for proper neuronal connectivity. Rho GTPases have been implicated in the regulation of dendrite development. However, the signaling pathways that impinge on these molecular switches remain poorly understood. Here we show that Wnt7b, which is expressed in the mouse hippocampus, increases dendritic branching in cultured hippocampal neurons. This effect is mimicked by the expression of Dishevelled (Dvl) and is blocked by Sfrp1, a secreted Wnt antagonist. Consistent with these findings, hippocampal neurons from mice lacking Dvl1 show reduced dendritic arborization. Activation of the canonical Wnt-Gsk3β pathway does not affect dendritic development. In contrast, Wnt7b and Dvl activate Rac and JNK in hippocampal neurons. Dominant-negative Rac, dominant-negative JNK or inhibition of JNK blocks Dvl-mediated dendritic growth. These findings demonstrate a new function for the non-canonical Wnt pathway in dendrite development and identify Dvl as a regulator of Rho GTPases and JNK during dendritic morphogenesis.

title: Nicotinic receptor subtypes in the developing chick brain: appearance of a species containing the alpha4, beta2, and alpha5 gene products.
abstract: Increasing evidence suggests nicotinic receptors regulate developmental events in the nervous system. We used [3H]epibatidine and 125I-alpha-bungarotoxin, together with subunit-specific monoclonal antibodies, to distinguish and quantify nicotinic receptor subtypes in developing chick brain. The results show that more than three fourths of the epibatidine-binding receptors at both early and late embryonic stages contain alpha4 and beta2 subunits, representing receptors previously distinguished by high affinity nicotine binding. A fraction of these also contain the alpha5 gene product, which is consistent with studies on transfected cells showing that the alpha4, beta2, and alpha5 gene products coassemble to produce epibatidine-binding receptors. A small portion of the receptors contain alpha3 and beta4 subunits, assembled in part with either alpha4 or beta2 subunits. The most abundant nicotinic receptors, however, at both early and late embryonic stages are those having high affinity for alpha-bungarotoxin rather than epibatidine. Most contain alpha7 subunits, whereas about half contain alpha8 subunits as well. The sharpest developmental increase between embryonic days 8 and 17/18 occurs with receptors containing alpha5 subunits, whereas receptors containing alpha3 or beta4 subunits undergo no specific increase. The three major receptor species (containing alpha4 and beta2 but not alpha5 subunits; alpha7 subunits; or alpha7 and alpha8 subunits) each increase approximately 3-fold during the same period. The results indicate greater receptor complexity than appreciated previously; they provide information about the rules governing subunit assembly in neuronal nicotinic receptors and draw attention to the role of alpha5 subunits in late development.

title: Wnt signaling function in Alzheimer’s disease
abstract: Alzheimer's disease (AD) is a neurodegenerative disease with progressive dementia accompanied by three main structural changes in the brain: diffuse loss of neurons; intracellular protein deposits termed neurofibrillary tangles (NFT) and extracellular protein deposits termed amyloid or senile plaques, surrounded by dystrophic neurites. Two major hypotheses have been proposed in order to explain the molecular hallmarks of the disease: The 'amyloid cascade' hypothesis and the 'neuronal cytoskeletal degeneration' hypothesis. While the former is supported by genetic studies of the early-onset familial forms of AD (FAD), the latter revolves around the observation in vivo that cytoskeletal changes - including the abnormal phosphorylation state of the microtubule associated protein tau - may precede the deposition of senile plaques. Recent studies have suggested that the trafficking process of membrane associated proteins is modulated by the FAD-linked presenilin (PS) proteins, and that amyloid beta-peptide deposition may be initiated intracellularly, through the secretory pathway. Current hypotheses concerning presenilin function are based upon its cellular localization and its putative interaction as macromolecular complexes with the cell-adhesion/signaling beta-catenin molecule and the glycogen synthase kinase 3beta (GSK-3beta) enzyme. Developmental studies have shown that PS proteins function as components in the Notch signal transduction cascade and that beta-catenin and GSK-3beta are transducers of the Wnt signaling pathway. Both pathways are thought to have an important role in brain development, and they have been connected through Dishevelled (Dvl) protein, a known transducer of the Wnt pathway. In addition to a review of the current state of research on the subject, we present a cell signaling model in which a sustained loss of function of Wnt signaling components would trigger a series of misrecognition events, determining the onset and development of AD.

title: Acetylcholinesterase promotes the aggregation of amyloid-beta-peptide fragments by forming a complex with the growing fibrils.
abstract: Acetylcholinesterase (AChE), an enzyme involved in the hydrolysis of the neurotransmitter acetylcholine, consistently colocalizes with the amyloid deposits characteristic of Alzheimer's disease and may contribute to the generation of amyloid proteins and/or physically affect fibril assembly. In order to identify the structural domains of the amyloid-beta-peptide (Abeta) involved in the aggregation induced by AChE, we have studied the effect of this cholinergic enzyme on Abeta peptide fragments of different sizes. AChE enhanced the aggregation of the Abeta(12-28) and Abeta(25-35) peptides but not of the Abeta(1-16) fragment. The inductive effect of AChE on the aggregation of Abeta(12-28) was abolished by the presence of either Abeta(1-16) or Abeta(9-21). The effect of the enzyme was also analysed using two different mutant fragments, possessing a low and the other a high capacity for fibrillogenesis. The fragments used were Abeta(12-28)Val18-->Ala and Abeta(12-28)Glu22-->Gln, respectively. AChE was able to promote the aggregation of these fragments in a very specific way and both mutant peptides were able to form amyloid fibrils, as revealed by negative staining under the electron microscope. Binding assays indicated that AChE was bound to Abeta(12-28), as well as to the Abeta(1-16) peptide. AChE was seen to form strong complexes with the Abeta(12-28) fibrils as such complexes stained positively for both thioflavine-T and AChE activity, were resistant to high ionic strength treatment, and were partially sensitive to detergents, suggesting that hydrophobic interactions may play a role in the stabilization of the AChE-Abeta complex. Our results suggest that such amyloid-AChE complexes are formed when AChE interacts with the growing amyloid fibrils and accelerates the assembly of Abeta peptides. This is consistent with the fact that AChE is known to be present within Abeta deposits including the pre-amyloid diffuse and mature senile plaques found in Alzheimer's brain.

title: Cellular mechanisms underlying the rhythmic bursts induced by NMDA microiontophoresis at the apical dendrites of CA1 pyramidal neurons
abstract: This article reports the cellular mechanisms underlying a form of intracellular “theta‐like” (θ‐like) rhythm evoked in vitro by microiontophoresis of N‐methyl‐D‐aspartate (NMDA) at the apical dendrites of CA1 pyramidal neurons. Rhythmic membrane potential (Vm) oscillations and action potential (AP) bursts (≈6 Hz; ≈20 mV; ≈2–5 APs) were evoked in all cells. The response lasted ≈2 s, and the initial oscillations were usually small (<20 mV) and below AP threshold. Rhythmic bursts were never evoked by imposed depolarization in the absence of NMDA. Block of Na+ conductance with tetrodotoxin (TTX) (1.5 μM), of non‐NMDA receptors with 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) (20 μM) and of synaptic inhibition by bicuculline (50 μM) and picrotoxin (50 μM) did not prevent NMDA oscillation. Inhibition of the voltage dependence of the NMDA conductance in Mg2+‐free Ringer's solution blocked oscillations. Preventing Ca2+ influx with Ca2+‐free and Co2+ (2‐mM) solutions and block of the slow Ca2+‐dependent afterhyperpolarization (sAHP) by carbamilcholine (5 μM), isoproterenol (10 μM), and intracellular BAPTA blocked NMDA oscillations. Inhibition of L‐type Ca2+ conductance with nifedipine (30 μM) reduced oscillation amplitude. Block of tetraethylammonium (TEA) (10 mM) and 4AP (10 mM)‐sensitive K+ conductance increased the duration and amplitude, but not the frequency, of oscillations. In conclusion, θ‐like bursts relied on the voltage dependence of the NMDA conductance and on high‐threshold Ca2+ spikes to initiate and boost the depolarizing phase of oscillations. The repolarization is initiated by TEA‐sensitive K+ conductance and is controlled by the sAHP. These results suggest a role of interactions between NMDA conductance and intrinsic membrane properties in generating the CA1 θ‐rhythm. Hippocampus 2003;13:10067.

title: The SAD-1 Kinase Regulates Presynaptic Vesicle Clustering and Axon Termination
abstract: During synapse formation, presynaptic axon outgrowth is terminated, presynaptic clusters of vesicles are associated with active zone proteins, and active zones are aligned with postsynaptic neurotransmitter receptors. We report here the identification of a novel serine/threonine kinase, SAD-1, that regulates several aspects of presynaptic differentiation in C. elegans. In sad-1 mutant animals presynaptic vesicle clusters in sensory neurons and motor neurons are diffuse and disorganized. Sensory axons fail to terminate in sad-1 mutants, whereas overexpression of SAD-1 causes sensory axons to terminate prematurely. SAD-1 protein is expressed in the nervous system and localizes to synapse-rich regions of the axons. SAD-1 is related to PAR-1, a kinase that regulates cell polarity during asymmetric cell division. Overexpression of SAD-1 causes mislocalization of vesicle proteins to dendrites, suggesting that sad-1 affects axonal-dendritic polarity as well as synaptic development.

title: Functional characteristics of unmyelinated fibres in the hippocampal cortex
abstract: (1) In transverse hippocampal slices (350 micrometer thick), taken from guinea pigs initially anaesthetized with ether, intracortical afferent fibres were activated by small current pulses delivered through tungsten microelectrodes. Extracellular potentials were recorded from the zone of activated fibres in dendritic layers while intracellular recordings were made from the soma of CA1 pyramidal cells. (2) When recording was made from the same level as the stimulating cathode, the extracellular potential consisted of a diphasic deflection followed by a larger negative wave with a superimposed population spike. The negative wave corresponded to an intracellularly recorded EPSP, and is called an extracellular EPSP, whereas the initial diphasic deflection had no intracellular counterpart. (3) The initial diphasic deflection was linearly related to the size of both the intracellular and extracellular EPSP. It was not changed by removal of calcium ions from the bathing fluid, whereas all postsynaptic activity disappeared. The diphasic deflection was propagated along fibres lying parallel to the pyramidal layer with a velocity of 0.3 m/sec. It could follow short bursts of stimulation at 300 Hz. The absolute refractory period was 2.0 msec. (4) The initial diphasic deflection is interpreted as the compound action potential of the largely unmyelinated afferent fibres to the CA1 neurones.