Chemical synapses are the predominant neuron-to-neuron contact in the central nervous system. Presynaptic boutons of neurons contain hundreds of vesicles filled with neurotransmitters, the diffusible signaling chemicals. Changes in the number of synapses are associated with numerous brain disorders, including Alzheimer's disease and epilepsy. However, all current approaches for measuring synaptic density in humans require brain tissue from autopsy or surgical resection. We report the use of the synaptic vesicle glycoprotein 2A (SV2A) radioligand [(11)C]UCB-J combined with positron emission tomography (PET) to quantify synaptic density in the living human brain. Validation studies in a baboon confirmed that SV2A is an alternative synaptic density marker to synaptophysin. First-in-human PET studies demonstrated that [(11)C]UCB-J had excellent imaging properties. Finally, we confirmed that PET imaging of SV2A was sensitive to synaptic loss in patients with temporal lobe epilepsy. Thus, [(11)C]UCB-J PET imaging is a promising approach for in vivo quantification of synaptic density with several potential applications in diagnosis and therapeutic monitoring of neurological and psychiatric disorders.

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Recovery from neuronal activation requires rapid clearance of potassium ions (K ؉ ) and restoration of osmotic equilibrium. The predominant water channel protein in brain, aquaporin-4 (AQP4), is concentrated in the astrocyte end-feet membranes adjacent to blood vessels in neocortex and cerebellum by association with ␣-syntrophin protein. Although AQP4 has been implicated in the pathogenesis of brain edema, its functions in normal brain physiology are uncertain. In this study, we used immunogold electron microscopy to compare hippocampus of WT and ␣-syntrophin-null mice (␣-Syn ؊/؊ ). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the ␣-Syn ؊/؊ mice, whereas labeling of the inwardly rectifying K ؉ channel, Kir4.1, is largely unchanged. Activity-dependent changes in K ؉ clearance were studied in hippocampal slices to test whether AQP4 and K ؉ channels work in concert to achieve isosmotic clearance of K ؉ after neuronal activation. Microelectrode recordings of extracellular K ؉ ([K ؉ ]o) from the target zones of Schaffer collaterals and perforant path were obtained after 5-, 10-, and 20-Hz orthodromic stimulations. K ؉ clearance was prolonged up to 2-fold in ␣-Syn ؊/؊ mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the ␣-Syn ؊/؊ mice. These studies lead us to propose that water flux through perivascular AQP4 is needed to sustain efficient removal of K ؉ after neuronal activation.

Background Clinical studies report that scopolamine, an acetylcholine muscarinic receptor antagonist, produces rapid antidepressant effects in depressed patients, but the mechanisms underlying the therapeutic response have not been determined. The present study examines the role of the mammalian target of rapamycin complex 1 (mTORC1) and synaptogenesis, which have been implicated in the rapid actions of NMDA receptor antagonists. Methods The influence of scopolamine on mTORC1 signaling was determined by analysis of the phosphorylated and activated forms of mTORC1 signaling proteins in the prefrontal cortex (PFC). The numbers and function of spine synapses were analyzed by whole cell patch clamp recording and 2-photon image analysis of PFC neurons. The actions of scopolamine were examined in the forced swim test in the absence or presence of selective mTORC1 and AMPA receptor inhibitors. Results The results demonstrate that a single, low dose of scopolamine rapidly increases mTORC1 signaling and the number and function of spine synapses in layer V pyramidal neurons in the PFC. Scopolamine administration also produces an antidepressant response in the forced swim test that is blocked by pretreatment with the mTORC1 inhibitor or by a glutamate AMPA receptor antagonist. Conclusions Taken together, the results demonstrate that the antidepressant actions of scopolamine require mTORC1 signaling and are associated with increased glutamate transmission, and synaptogenesis, similar to NMDA receptor antagonists. These findings provide novel targets for safer and more efficacious rapid acting antidepressant agents.

We recently described a pronounced neuronal loss in layer III of the entorhinal cortex (EC) in patients with intractable temporal lobe epilepsy (Du et al., 1993a). To explore the pathophysiology underlying this distinct neuropathology, we examined the EC in three established rat models of epilepsy using Nissl staining and parvalbumin immunohistochemistry. Adult male rats were either electrically stimulated in the ventral hippocampus for 90 min or injected with kainic acid or lithium/pilocarpine. Animals were observed for behavioral changes for up to 6 hr and were killed 24 hr or 4 weeks after the experimental treatments. At 24 hr, all animals that had exhibited a bout of acute status epilepticus showed a consistent pattern of neuronal loss in the EC in Nissl-stained sections. Neurodegeneration was most pronounced in layer III of the medial Ec at all dorsoventral levels. A few surviving neurons were frequently present in the lesioned area. An identical pattern of nerve cell loss was also seen in the EC of rats killed 4 weeks following the treatments. This lesion was completely prevented by an injection of diazepam and pentobarbital, given 1 hr after kainic acid administration. Immunohistochemistry demonstrated a relative resistance of parvalbumin-positive neurons in layer III of the medial EC. Taken together, these experiments indicate that prolonged seizures cause a preferential neuronal loss in layer III of the medial EC and that this lesion may be related to a pathological elevation of intracellular calcium ion concentrations.

An abnormal accumulation of extracellular K ؉ in the brain has been implicated in the generation of seizures in patients with mesial temporal lobe epilepsy (MTLE) and hippocampal sclerosis. Experimental studies have shown that clearance of extracellular K ؉ is compromised by removal of the perivascular pool of the water channel aquaporin 4 (AQP4), suggesting that an efficient clearance of K ؉ depends on a concomitant water flux through astrocyte membranes. Therefore, we hypothesized that loss of perivascular AQP4 might be involved in the pathogenesis of MTLE. Whereas Western blot analysis showed an overall increase in AQP4 levels in MTLE compared with non-MTLE hippocampi, quantitative ImmunoGold electron microscopy revealed that the density of AQP4 along the perivascular membrane domain of astrocytes was reduced by 44% in area CA1 of MTLE vs. non-MTLE hippocampi. There was no difference in the density of AQP4 on the astrocyte membrane facing the neuropil. Because anchoring of AQP4 to the perivascular astrocyte endfoot membrane depends on the dystrophin complex, the localization of the 71-kDa brain-specific isoform of dystrophin was assessed by immunohistochemistry. In non-MTLE hippocampus, dystrophin was preferentially localized near blood vessels. However, in the MTLE hippocampus, the perivascular dystrophin was absent in sclerotic areas, suggesting that the loss of perivascular AQP4 is secondary to a disruption of the dystrophin complex. We postulate that the loss of perivascular AQP4 in MTLE is likely to result in a perturbed flux of water through astrocytes leading to an impaired buffering of extracellular K ؉ and an increased propensity for seizures.dystrophin ͉ epilepsy ͉ seizures ͉ astrocytes M esial temporal lobe epilepsy (MTLE) is one of the commonest forms of medically intractable epilepsies. MTLE is characterized by seizures that originate from mediobasal temporal lobe structures, particularly the hippocampus, and neurosurgical resection of the epileptogenic hippocampus is often used to treat this disorder. The resected, epileptogenic hippocampus in MTLE is typically indurated and atrophic and displays massive loss of neurons along with astroglial changes, particularly in areas CA1 and CA3 and the dentate hilus, a condition known as hippocampal (or Ammon's horn) sclerosis. Electrophysiological recordings from MTLE hippocampi have demonstrated that these hippocampi are hyperexcitable when compared with nonsclerotic hippocampi from patients with other types of temporal lobe epilepsy, such as mass associated temporal lobe epilepsy (patients with an extrahippocampal mass lesion) or paradoxical temporal lobe epilepsy (patients without a mass lesion and with seizures of unknown etiology). A fundamental question that remains to be resolved is why the MTLE hippocampus is hyperexcitable.Studies of MTLE patient hippocampi have shown that the K ϩ buffering capacity is diminished when compared with non-

Astrocytes play a critical role in regulation of extracellular neurotransmitter levels in the central nervous system. This function is particularly prominent for the excitatory amino acid glutamate, with estimates that 80–90% of extracellular glutamate uptake in brain is through astrocytic glutamate transporters. This uptake has significance both in regulation of the potential toxic accumulation of extracellular glutamate, and in normal resupply of inhibitory and excitatory synapses with neurotransmitter. This resupply of neurotransmitter is accomplished by astroglial uptake of glutamate, transformation of glutamate to glutamine by the astrocytic enzyme glutamine synthetase, and shuttling of glutamine back to excitatory and inhibitory neurons via specialized transporters. Once in neurons, glutamine is enzymatically converted back to glutamate, which is utilized for synaptic transmission, either directly, or following decarboxylation to GABA. Many neurologic and psychiatric conditions, particularly epilepsy, are accompanied by the development of reactive gliosis, a pathology characterized by anatomical and biochemical plasticity in astrocytes, accompanied by proliferation of these cells. Among the biochemical changes evident in reactive astrocytes is a downregulation of several of the important regulators of the glutamine-glutamate cycle, including glutamine synthetase, and possibly also glutamate transporters. This downregulation may have significance in contributing both to the aberrant excitability and to the altered neuropathology characterizing epilepsy. In the present review, we provide an overview of the normal function of astrocytes in regulating extracellular glutamate homeostasis, neurotransmitter supply, and excitotoxicity. We further discuss the potential role reactive gliosis may play in the pathophysiology of epilepsy.

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