Overexpression of the amyloid precursor protein (APP) in hippocampal neurons leads to elevated ␤-amyloid peptide (A␤) production and consequent depression of excitatory transmission. The precise mechanisms underlying APP-induced synaptic depression are poorly understood. Uncovering these mechanisms could provide insight into how neuronal function is compromised before cell death during the early stages of Alzheimer's disease. Here we verify that APP up-regulation leads to depression of transmission in cultured hippocampal autapses; and we perform whole-cell recording, FM imaging, and immunocytochemistry to identify the specific mechanisms accounting for this depression. We find that APP overexpression leads to postsynaptic silencing through a selective reduction of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated currents. This effect is likely mediated by A␤ because expression of mutant APP incapable of producing A␤ did not depress transmission. In addition, although we eliminate presynaptic silencing as a mechanism underlying APP-mediated inhibition of transmission, we did observe an A␤-induced presynaptic deficit in vesicle recycling with sustained stimulation. These findings demonstrate that APP elevation disrupts both presynaptic and postsynaptic compartments.Alzheimer's disease ͉ synaptic transmission ͉ glutamate receptor ͉ synaptic vesicle cycling T he early stages of Alzheimer's disease (AD) are characterized by cognitive deficits that are likely the result of impairments of synaptic function before either plaque formation or cell death (1-4). Identifying these impairments and the mechanisms that underlie them is a critical step in the development of novel therapies aimed at improving cognitive function in AD patients.A critical role for amyloid precursor protein (APP) and its cleavage products is indicated by the finding that mutations in the APP gene are linked to the inherited form of the disease, familial early onset AD (FAD; ref. 5). In addition, animal models that express the human APP protein bearing FAD-linked mutations recapitulate key features of early stage AD pathology (6, 7). Notably, levels of the ␤-amyloid peptide (A␤), which is generated through sequential cleavage of APP by ␤-APPcleaving enzyme 1 (BACE) and ␥-secretase, are greatly elevated in these model mice (7).Three lines of evidence strongly suggest that A␤ has acute effects on synaptic function: (i) Intracerebroventricular injection of naturally secreted A␤ is sufficient to disrupt cognitive function in a rapid and transient manner (8).(ii) Active immunization with A␤ peptide can protect against, or even reverse, AD-like neuropathology and behavioral deficits of AD model mice (9, 10). (iii) Passive immunization with anti-A␤ antibody can rapidly reverse memory deficits of AD model mice (11-13).Consistent with this notion, rapid viral-mediated upregulation of APP and consequent elevation of A␤ production in cultured hippocampal slices has been shown to depress excitatory transmission by decreasing the num...

Prolonged exposure to cannabinoids results in tolerance in vivo and desensitization of cannabinoid receptors in vitro. We show here that cannabinoid-induced presynaptic inhibition of glutamatergic neurotransmission desensitized after prolonged exposure to the cannabinoid receptor agonist ( Win55,. Synaptic activity between hippocampal neurons in culture was determined from network-driven increases in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i spikes) and excitatory postsynaptic currents. Win55,212-2-induced (100 nM) inhibition partially desensitized after 2 h and completely desensitized after 18-to 24-h exposure. The desensitization could be overcome by higher concentrations of agonist as indicated by a parallel rightward shift of the concentration response curve from an EC 50 of 2.7 Ϯ 0.3 nM to 320 Ϯ 147 nM for inhibition of [Ca 2ϩ ] i spiking and from 43 Ϯ 17 nM to 4505 Ϯ 403 nM for inhibition of synaptic currents, suggesting that this phenomenon may underlie tolerance. Presynaptic expression of dominant negative G-protein-coupled-receptor kinase (GRK2-Lys220Arg) or ␤-arrestin (319 -418) reduced the desensitization produced by 18-to 24-h pretreatment with 100 nM, Win55,212-2 suggesting that desensitization followed the prototypical pathway for G-protein-coupled receptors. Prolonged treatment with Win55,212-2 produced a modest increase in the EC 50 for adenosine inhibition of synaptic transmission and pretreatment with cyclopentyladenosine produced a slight increase in the EC 50 for Win55,212-2, suggesting a reciprocal ability to produce heterologous desensitization. The long-term changes in synaptic function that accompany chronic cannabinoid exposure will be an important factor in evaluating the therapeutic potential of these drugs and will provide insight into the role of the endocannabinoid system.

The therapeutic use of progesterone following traumatic brain injury has recently entered phase III clinical trials as a means of neuroprotection. Although it has been hypothesized that progesterone protects against calcium overload following excitotoxic shock, the exact mechanisms underlying the beneficial effects of progesterone have yet to be determined. We found that therapeutic concentrations of progesterone to be neuroprotective against depolarization-induced excitotoxicity in cultured striatal neurons. Through use of calcium imaging, electrophysiology and the measurement of changes in activity-dependent gene expression, progesterone was found to block calcium entry through voltage-gated calcium channels, leading to alterations in the signaling of the activity-dependent transcription factors NFAT and CREB. The effects of progesterone were highly specific to this steroid hormone, although they did not appear to be receptor mediated. In addition, progesterone did not inhibit AMPA or NMDA receptor signaling. This analysis regarding the effect of progesterone on calcium signaling provides both a putative mechanism by which progesterone acts as a neuroprotectant, as well as affords a greater appreciation for its potential far-reaching effects on cellular function.

Synaptotagmin IV (Syt IV) is a brain-specific isoform of the synaptotagmin family, the levels of which are strongly elevated after seizure activity. The dominant hypothesis of Syt IV function states that Syt IV upregulation is a neuroprotective mechanism for reducing neurotransmitter release. To test this hypothesis in mammalian CNS synapses, Syt IV was overexpressed in cultured mouse hippocampal neurons, and acute effects on fast excitatory neurotransmission were assessed. We found neurotransmission unaltered with respect to basal release probability, Ca 2ϩ dependence of release, short-term plasticity, and fusion pore kinetics. In contrast, expression of a mutant Syt I with diminished Ca 2ϩ affinity (R233Q) reduced release probability and altered the Ca 2ϩ dependence of release, thus demonstrating the sensitivity of the system to changes in neurotransmission resulting from changes to the Ca 2ϩ sensor. Together, these data refute the dominant model that Syt IV functions as an inhibitor of neurotransmitter release in mammalian neurons.

The administration of progesterone as a neuroprotective agent following traumatic brain injury has recently entered phase III clinical trials. Previous work has demonstrated that therapeutic concentrations of progesterone decrease excitotoxicity through direct inhibition of voltage-gated calcium channels, an action independent of the nuclear progesterone receptor. Here we report using cultured rat striatal neurons that these same concentrations of progesterone also block voltage-gated potassium channels, sodium channels and GABAA currents. The actions of progesterone act at the surface membrane of neurons in a steroid specific, voltage-independent, concentration-dependent manner. Notably, these broad actions of progesterone on ion channel and neurotransmitter receptor function mirror those of dihydropyridines, and indicate potential shared mechanisms of action, the prospective of additional therapeutic applications, and possibly, untoward effects.