Steven Mennerick

Synaptic Activity Regulates Interstitial Fluid Amyloid-β Levels In Vivo

Summary Aggregation of the amyloid-β (Aβ) peptide in the extracellular space of the brain is central to Alzheimers disease pathogenesis. Aβ aggregation is concentration dependent and brain region specific. Utilizing in vivo microdialysis concurrently with field potential recordings, we demonstrate that Aβ levels in the brain interstitial fluid are dynamically and directly influenced by synaptic activity on a timescale of minutes to hours. Using an acute brain slice model, we show that the rapid effects of synaptic activity on Aβ levels are primarily related to synaptic vesicle exocytosis. These results suggest that synaptic activity may modulate a neurodegenerative disease process, in this case by influencing Aβ metabolism and ultimately region-specific Aβ deposition. The findings also have important implications for treatment development.

Endocytosis is required for synaptic activity-dependent release of amyloid-β in vivo

Aggregation of amyloid-beta (Abeta) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimers disease. Full-length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Abeta. Abeta is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Abeta generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Abeta levels in vivo. Two distinct methods that increased synaptic transmission resulted in an elevation of ISF Abeta levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Abeta. We estimate that approximately 70% of ISF Abeta arises from endocytosis-associated mechanisms, with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention.

Ultrafast Exocytosis Elicited by Calcium Current in Synaptic Terminals of Retinal Bipolar Neurons

Using high resolution capacitance measurements, we have characterized an ultrafast component of transmitter release in ribbon-type synaptic terminals of retinal bipolar neurons. During depolarization, capacitance increases to a plateau of approximately 30 fF with a time constant of approximately 1.5 ms. When not limited by activation kinetics of calcium current, the small pool is depleted even faster, with a time constant of 0.5 ms. After the ultrafast pool is depleted, capacitance rises with a slower time constant of approximately 300 ms. EGTA (5 mM) depresses the slower capacitance rise but leaves the ultrafast phase intact. BAPTA (5 mM) depresses both components of exocytosis. With paired-pulse stimulation, the ultrafast pool recovers from depletion with a time constant of approximately 4 s. The ultrafast component may represent fusion of docked vesicles at the base of the synaptic ribbon, while the slower component represents more distal vesicles on the ribbon.

Paired-pulse modulation of fast excitatory synaptic currents in microcultures of rat hippocampal neurons.

1. Paired‐pulse modulation of excitatory non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor‐mediated autaptic currents and conventional monosynaptic (interneuronal) excitatory postsynaptic currents (EPSCs) was investigated in microcultures of rat hippocampal neurons, where polysynaptic influences are eliminated. 2. Most autaptic currents and EPSCs exhibited paired‐pulse depression in response to paired stimuli. Depression was sensitive to the level of transmitter release, which was varied by manipulating extracellular Ca2+ and Mg2+ concentrations. Paired‐pulse facilitation emerged in many cells at low levels of transmitter release. 3. Paired‐pulse depression and facilitation could be differentially expressed at two distinct postsynaptic targets of a single presynaptic cell, and the form of modulation was not dependent upon the transmitter phenotype of the postsynaptic cell. 4. Paired‐pulse depression recovered exponentially with a time constant of approximately 5 s, although in most neurons a much faster component of recovery was detected. Recovery from paired‐pulse facilitation was well described by a single exponential of 380 +/‐ 57 ms. 5. Under conditions of robust paired‐pulse depression of evoked responses, spontaneous autaptic and postsynaptic currents (sEPSCs, presumed miniature EPSCs) occurred at an enhanced frequency immediately following evoked responses. The decay of the frequency increase mirrored the time course of recovery from paired‐pulse facilitation of evoked responses examined under conditions of reduced transmitter release. 6. Several lines of evidence suggested a large presynaptic component to paired‐pulse depression. In eight out of nine cells no depression in sEPSC amplitudes was detected following conditioning stimulation. Simultaneously recorded glial glutamate uptake currents showed depression similar to neuronal evoked EPSCs. Finally, NMDA receptor‐mediated EPSC paired‐pulse depression at positive potentials was similar to non‐NMDA EPSC depression. 7. Neither adenosine nor glutamate feedback onto presynaptic receptors is likely to mediate paired‐pulse depression, because neither competitive nor non‐competitive inhibitors of the actions of these agents diminished paired‐pulse depression.

Impaired glial glutamate transport in a mouse tuberous sclerosis epilepsy model

Excessive astrocytosis in cortical tubers in tuberous sclerosis complex (TSC) suggests that astrocytes may be important for epileptogenesis in TSC. We previously demonstrated that astrocyte‐specific Tsc1 gene inactivation in mice (Tsc1 cKO mice) results in progressive epilepsy. Here, we report that glutamate transporter expression and function is impaired in Tsc1 cKO astrocytes. Tsc1 cKO mice exhibit decreased GLT‐1 and GLAST protein expression. Electrophysiological assays demonstrate a functional decrease in glutamate transport currents of Tsc1 cKO astrocytes in hippocampal slices and astrocyte cultures. These findings suggest that Tsc1 inactivation in astrocytes causes dysfunctional glutamate homeostasis, leading to seizure development in TSC. Ann Neurol 2003

Neurosteroid Access to the GABAA Receptor

GABAA receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABAA receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABAA receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

Reluctant Vesicles Contribute to the Total Readily Releasable Pool in Glutamatergic Hippocampal Neurons

The size of the readily releasable pool (RRP) of vesicles is critically important for determining the size of postsynaptic currents generated in response to action potentials. However, discrepancies in RRP estimates exist among methods designed to measure RRP size. In glutamatergic hippocampal neurons, we found that hypertonic sucrose application yielded RRP size estimates approximately fivefold larger than values obtained with high-frequency action potential trains commonly assumed to deplete the RRP. This discrepancy was specific for glutamatergic neurons, because no difference was found between sucrose and train estimates of RRP size in GABAergic neurons. A small component of the difference in excitatory neurons was accounted for by postsynaptic receptor saturation. Train estimates of vesicle pool size obtained using more stimuli revealed that action potential-elicited EPSCs did not truly reach a steady state during shorter trains, and RRP estimates were closer to sucrose estimates made in the same neurons. This suggested that reluctant vesicles may contribute to the total available pool. Two additional lines of evidence supported this hypothesis. First, RRP estimates from strongly depolarizing hyperkalemic solutions closely matched those obtained with sucrose. Second, when Ca2+ influx was enhanced during trains, train estimates of pool size matched those obtained with sucrose. These data suggest that glutamatergic hippocampal neurons maintain a heterogeneous population of vesicles that can be differentially released with varying Ca2+ influx, thereby increasing the range of potential synaptic responses.

Covalent and Noncovalent Interactions Mediate Metabotropic Glutamate Receptor mGlu5 Dimerization

Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity similar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.

Neuronal Expression of the Glutamate Transporter GLT-1 in Hippocampal Microcultures

To address the question of the relative contributions of glial and neuronal glutamate transport in the vertebrate CNS, we studied the distribution of forebrain glutamate transporters in rat hippocampal microcultures, a preparation in which physiological functions of glutamate transporters have been well characterized. Two of the three transporters, GLAST (EAAT1) and EAAC1 (EAAT3), are localized to microculture glia and neurons, respectively, as expected. However, we find strong immunoreactivity for the third glutamate transporter GLT-1 (EAAT2), a putatively glial transporter, in microculture neurons and in a small subset of microculture glia. Indistinguishable immunohistochemical staining patterns for GLT-1 were obtained with antibodies directed against both the N terminal and C terminal of the GLT-1 protein. Double-labeling experiments suggest that neuronal GLT-1 protein is primarily localized to the dendrites of excitatory neurons. Neuronal electrogenic transport currents in response tod-aspartate applications were occluded by the selective GLT-1 inhibitor dihydrokainate. In contrast, glia exhibited a larger transporter current density than did neurons, and the glial transport current was less sensitive to dihydrokainate. Neuronal transport currents were potentiated less than were glial currents when the chaotropic anion thiocyanate was substituted for gluconate in the whole-cell recording pipette, consistent with the previously reported lower anion permeability of EAAC1 and GLT-1 compared with that of GLAST. After microculture glia were rendered nonviable, excitatory autaptic currents (EACs) were prolonged in the presence of dihydrokainate, suggesting that neuronal GLT-1 is capable of participating in the clearance of synaptically released glutamate. Our results suggest that the initially proposed characterization of GLT-1 as a purely glial transporter is too simplistic and that under certain conditions functional GLT-1 protein can be expressed in brain neurons. The study suggests that changes in GLT-1 levels that occur with pathology or experimental manipulations cannot be assumed to be glial.

New evidence that both T-type calcium channels and GABAA channels are responsible for the potent peripheral analgesic effects of 5α-reduced neuroactive steroids

&NA; Neurosteroids are potent blockers of neuronal low‐voltage activated (T‐type) Ca2+ channels and potentiators of GABAA ligand‐gated channels, but their effects in peripheral pain pathways have not been studied previously. To investigate potential analgesic effects and the ion channels involved, we tested the ability of locally injected 5α‐reduced neurosteroids to modulate peripheral thermal nociception to radiant heat in adult rats in vivo and to modulate GABAA and T‐type Ca2+ channels in vitro. The steroid anesthetic alphaxalone (ALPX), the endogenous neurosteroid allopregnanolone (3α5αP), and a related compound ((3α,5α,17β)‐3‐hydroxyandrostane‐17‐carbonitrile, (ACN)), induced potent, dose‐dependent, enantioselective anti‐nociception in vivo and modulation of both T‐type Ca2+ currents and GABAA‐mediated currents in vitro. Analgesic effects of ALPX were incompletely antagonized by co‐injections of the GABAA receptor antagonist bicuculline. The neurosteroid analogue ((3α,5α)‐3‐hydroxy‐13,24‐cyclo‐18,21‐dinorchol‐22‐en‐24‐ol (CDNC24), a compound with GABAergic but not T‐type activity, was not analgesic. However, (3β,5α,17β)‐17‐hydroxyestrane‐3‐carbonitrile (ECN)), which has effects on T‐type channels but not on GABAA receptors, also induced potent enantioselective peripheral anti‐nociception. ECN increased pain thresholds less than ALPX, 3α5αP and ACN. However, when an ineffective dose of CDNC24 was combined with ECN, anti‐nociceptive activity was greatly enhanced, and this effect was bicuculline‐sensitive. These results strongly suggest that GABAA channels do not contribute to baseline pain transmission, but they can enhance anti‐nociception mediated by blockade of T‐type Ca2+ channels. In conclusion, we demonstrate that potent peripheral analgesia induced by 5α‐reduced neurosteroid is mediated in part by effects on T‐type Ca2+ channels. Our results also reveal a role of GABA‐gated ion channels in peripheral nociceptive signaling.