Regulation of dark period sleep by the Amygdala: A microinjection and optogenetics study.

The central nucleus of the amygdala (CNA) projects to brainstem regions that generate and regulate rapid eye movement sleep (REM). We used optogenetics to assess the influence of CNA inputs into reticularis pontis oralis (RPO), pedunculopontine tegmentum (PPT) and nucleus subcoeruleus (SubC) on dark period sleep. We compared these results to effects of microinjections into CNA of the GABAA agonist, muscimol (MUS, inhibition of cell bodies) and tetrodotoxin (TTX, inhibition of cell bodies and fibers of passage). For optogenetics, male Wistar rats received excitatory (AAV5-EF1a-DIO -hChR2(H134R)-EYFP) or inhibitory (AAV-EF1a-DIO-eNpHR3.0-EYFP; DIO-eNpHR3.0) opsins into CNA and AAV5-EF1a-mCherry-IRES-WGA-Cre into RPO, PPT, or SubC. This enabled only CNA neurons synaptically connected to each region to express opsin. Optic cannulae for light delivery into CNA and electrodes for determining sleep were implanted. Sleep was recorded with and without blue or amber light stimulation of CNA. Separate rats received MUS or TTX into CNA prior to recording sleep. Optogenetic activation of CNA neurons projecting to RPO enhanced REM and did not alter non-REM (NREM) whereas activation of CNA neurons projecting to PPT or SubC did not significantly affect sleep. Inhibition of CNA neurons projecting to any region did not significantly alter sleep. TTX inactivation of CNA decreased REM and increased NREM whereas muscimol inactivation did not significantly alter sleep. Thus, the amygdala can regulate decreases and increases in REM, and RPO is important for CNA promotion of REM. Fibers passing through CNA, likely from the basolateral nucleus of the amygdala, also play a role in regulating sleep.

Effects of stressor controllability on transcriptional levels of c-fos, Arc, and brain-derived neurotrophic factor in mouse amygdala and medial prefrontal cortex.

Controllability is an important factor in determining stress outcomes. Uncontrollable stress is associated with the development of psychopathology such as post-traumatic stress disorder, whereas controllable stress is associated with adaptive stress responses and positive outcomes. In this study, we investigated how controllability affects poststress neurobiology by assessing transcriptional levels of activity-dependent genes in medial prefrontal cortex (mPFC) and amygdala, regions important in mediating stress outcomes. Mice were subjected to either escapable shock (ES) or yoked inescapable shock (IS) as models of controllable and uncontrollable stress, respectively. Immediately (0 h) or at 2 h after shock training (20 trials; 0.5 mA, 5.0 s maximum duration; 1.0 min interstimulus interval), mice were killed, and we interrogated expression levels of the immediate-early genes, c-fos and Arc, and a delayed primary response gene, brain-derived neurotrophic factor, in mPFC, amygdala, and somatosensory cortex (a control region), using real-time reverse transcription quantitative PCR (RT qPCR). We found ES-associated up-regulation of brain-derived neurotrophic factor in amygdala as well as in mPFC. IS suppressed c-fos in mPFC (0 h) but induced more Arc in amygdala (2 h) in comparison with ES. Freezing, an index of fear memory, and serum level corticosterone, an index of the stress response, did not differ between mice trained with ES or IS. The data are discussed with respect to the potential functional involvements of the amygdala and mPFC in mediating differential outcomes of controllable and uncontrollable stress.

Effects of Optogenetic inhibition of BLA on Sleep Brief Optogenetic Inhibition of the Basolateral Amygdala in Mice Alters Effects of Stressful Experiences on Rapid Eye Movement Sleep.

Study Objectives: Stressful events can directly produce significant alterations in subsequent sleep, in particular rapid eye movement sleep (REM); however, the neural mechanisms underlying the process are not fully known. Here, we investigated the role of the basolateral nuclei of the amygdala (BLA) in regulating the effects of stressful experience on sleep. Methods: We used optogenetics to briefly inhibit glutamatergic cells in BLA during the presentation of inescapable footshock (IS) and assessed effects on sleep, the acute stress response, and fear memory. c-Fos expression was also assessed in the amygdala and the medial prefrontal cortex (mPFC), both regions involved in coping with stress, and in brain stem regions implicated in the regulation of REM. Results: Compared to control mice, peri-shock inhibition of BLA attenuated an immediate reduction in REM after IS and produced a significant overall increase in REM. Moreover, upon exposure to the shock context alone, mice receiving peri-shock inhibition of BLA during training showed increased REM without altered freezing (an index of fear memory) or stress-induced hyperthermia (an index of acute stress response). Inhibition of BLA during REM under freely sleeping conditions enhanced REM only when body temperature was high, suggesting the effect was influenced by stress. Peri-shock inhibition of BLA also led to elevated c-Fos expression in the central nucleus of the amygdala and mPFC and differentially altered c-Fos activity in the selected brain stem regions. Conclusions: Glutamatergic cells in BLA can modulate the effects of stress on REM and can mediate effects of fear memory on sleep that can be independent of behavioral fear.

Changes in the Hippocampal Proteome Associated with Spatial Memory Impairment after Exposure to Low (20 cGy) Doses of 1 GeV/n 56Fe Radiation.

Exposure to low (∼20 cGy) doses of high-energy charged (HZE) particles, such as 1 GeV/n 56Fe, results in impaired hippocampal-dependent learning and memory (e.g., novel object recognition and spatial memory) in rodents. While these findings raise the possibility that astronauts on deep-space missions may develop cognitive deficits, not all rats develop HZE-induced cognitive impairments, even after exposure to high (200 cGy) HZE doses. The reasons for this differential sensitivity in some animals that develop HZE-induced cognitive failure remain speculative. We employed a robust quantitative mass spectrometry-based workflow, which links early-stage discovery to next-stage quantitative verification, to identify differentially active proteins/pathways in rats that developed spatial memory impairment at three months after exposure to 20 cGy of 1 GeV/n 56Fe (20/impaired), and in those rats that managed to maintain normal cognitive performance (20/functional). Quantitative data were obtained on 665-828 hippocampal proteins in the various cohorts of rats studied, of which 580 were expressed in all groups. A total of 107 proteins were upregulated in the irradiated rats irrespective of their spatial memory performance status, which included proteins involved in oxidative damage response, calcium transport and signaling. Thirty percent (37/107) of these "radiation biomarkers" formed a functional interactome of the proteasome and the COP9 signalosome. These data suggest that there is persistent oxidative stress, ongoing autophagy and altered synaptic plasticity in the irradiated hippocampus, irrespective of the spatial memory performance status, suggesting that the ultimate phenotype may be determined by how well the hippocampal neurons compensate to the ongoing oxidative stress and associated side effects. There were 67 proteins with expression that correlated with impaired spatial memory performance. Several of the "impaired biomarkers" have been implicated in poor spatial memory performance, neurodegeneration, neuronal loss or neuronal susceptibility to apoptosis, or neuronal synaptic or structural plasticity. Therefore, in addition to the baseline oxidative stress and altered adenosine metabolism observed in all irradiated rats, the 20/impaired rats expressed proteins that led to poor spatial memory performance, enhanced neuronal loss and apoptosis, changes in synaptic plasticity and dendritic remodeling. A total of 46 proteins, which were differentially upregulated in the sham-irradiated and 20/functional rat cohorts, can thus be considered as markers of good spatial memory, while another 95 proteins are associated with the maintenance of good spatial memory in the 20/functional rats. The loss or downregulation of these "good spatial memory" proteins would most likely exacerbate the situation in the 20/impaired rats, having a major impact on their neurocognitive status, given that many of those proteins play an important role in neuronal homeostasis and function. Our large-scale comprehensive proteomic analysis has provided some insight into the processes that are altered after exposure, and the collective data suggests that there are multiple problems with the functionality of the neurons and astrocytes in the irradiated hippocampi, which appear to be further exacerbated in the rats that have impaired spatial memory performance or partially compensated for in the rats with good spatial memory.

Propofol-induced Inhibition of Catecholamine Release Is Reversed by Maintaining Calcium Influx.

BACKGROUND: Propofol (2,6-diisopropylphenol) is one of the most frequently used anesthetic agents. One of the main side effects of propofol is to reduce blood pressure, which is thought to occur by inhibiting the release of catecholamines from sympathetic neurons. Here, the authors hypothesized that propofol-induced hypotension is not simply the result of suppression of the release mechanisms for catecholamines. METHODS: The authors simultaneously compared the effects of propofol on the release of norepinephrine triggered by high K-induced depolarization, as well as ionomycin, by using neuroendocrine PC12 cells and synaptosomes. Ionomycin, a Ca ionophore, directly induces Ca influx, thus bypassing the effect of ion channel modulation by propofol. RESULTS: Propofol decreased depolarization (high K)-triggered norepinephrine release, whereas it increased ionomycin-triggered release from both PC12 cells and synaptosomes. The propofol (30 µM)-induced increase in norepinephrine release triggered by ionomycin was dependent on both the presence and the concentration of extracellular Ca (0.3 to 10 mM; n = 6). The enhancement of norepinephrine release by propofol was observed in all tested concentrations of ionomycin (0.1 to 5 µM; n = 6). CONCLUSIONS: Propofol at clinically relevant concentrations promotes the catecholamine release as long as Ca influx is supported. This unexpected finding will allow for a better understanding in preventing propofol-induced hypotension.

Exposure to mission relevant doses of 1 GeV/Nucleon (56)Fe particles leads to impairment of attentional set-shifting performance in socially mature rats.

Previous ground-based experiments have shown that cranial irradiation with mission relevant (20 cGy) doses of 1 GeV/nucleon (56)Fe particles leads to a significant impairment in Attentional Set Shifting (ATSET) performance, a measure of executive function, in juvenile Wistar rats. However, the use of head only radiation exposure and the biological age of the rats used in that study may not be pertinent to determine the likelihood that ATSET will be impaired in Astronauts on deep space flights. In this study we have determined the impact that whole-body exposure to 10, 15 and 20 cGy of 1 GeV/nucleon (56)Fe particles had on the ability (at three months post exposure) of socially mature (retired breeder) Wistar rats to conduct the attentional set-shifting paradigm. The current study has established that whole-body exposures to 15 and 20 (but not 10) cGy of 1 GeV/nucleon (56)Fe particles results in the impairment of ATSET in both juvenile and socially mature rats. However, the exact nature of the impaired ATSET performance varied depending upon the age of the rats, whether whole-body versus cranial irradiation was used and the dose of 1 GeV/u (56)Fe received. Exposure of juvenile rats to 20 cGy of 1 GeV/nucleon (56)Fe particles led to a decreased ability to perform intra-dimensional shifting (IDS) irrespective of whether the rats received head only or whole-body exposures. Juvenile rats that received whole-body exposure also had a reduced ability to habituate to the assay and to complete intra-dimensional shifting reversal (IDR), whereas juvenile rats that received head only exposure had a reduced ability to complete compound discrimination reversal (CDR). Socially mature rats that received whole-body exposures to 10 cGy of 1 GeV/nucleon (56)Fe particles exhibited no obvious decline in set-shifting performance; however those exposed to 15 and 20 cGy had a reduced ability to perform simple discrimination (SD) and compound discrimination (CD). Exposure to 20 cGy of 1 GeV/nucleon (56)Fe particles also led to a decreased performance in IDR and to ∼25% of rats failing to habituate to the task. Most of these rats started to dig for the food reward but rapidly (within 15 s) gave up digging, suggesting that they had developed appropriate procedural memories about food retrieval, but had an inability to maintain attention on the task. Our preliminary data suggests that whole-body exposure to 20 cGy of 1 GeV/nucleon (56)Fe particles reduced the cholinergic (but not the GABAergic) readily releasable pool (RRP) in nerve terminals of the basal forebrain from socially-mature rats. This perturbation of the cholinergic RRP could directly lead to the loss of CDR and IDR performance, and indirectly [through the metabolic changes in the medial prefrontal cortex (mPFC)] to the loss of SD and CD performance. These findings provide the first evidence that attentional set-shifting performance in socially mature rats is impaired after whole-body exposure to mission relevant doses (15 and 20 cGy) of 1 GeV/nucleon (56)Fe particles, and importantly that a dose reduction down to 10 cGy prevents that impairment. The ability to conduct Discrimination tasks (SD and CD) and reversal learning (CDR) is reduced after exposure to 15 and 20 cGy of 1 GeV/nucleon (56)Fe particles, but at 20 cGy there is an additional decrement, ∼ 25% of rats are unable to maintain attention to task. These behavioral decrements are associated with a reduction in the cholinergic RRP within basal forebrain, which has been shown to play a major role in regulating the activity of the PFC.

Executive function in rats is impaired by low (20 cGy) doses of 1 GeV/u (56)Fe particles.

Exposure to galactic cosmic radiation is a potential health risk in long-term space travel and represents a significant risk to the central nervous system. The most harmful component of galactic cosmic radiation is the HZE [high mass, highly charged (Z), high energy] particles, e.g., (56)Fe particle. In previous ground-based experiments, exposure to doses of HZE-particle radiation that an astronaut will receive on a deep space mission (i.e., ∼20 cGy) resulted in pronounced deficits in hippocampus-dependent learning and memory in rodents. Neurocognitive tasks that are dependent upon other regions of the brain, such as the striatum, are also impaired after exposure to low HZE-particle doses. These data raise the possibility that neurocognitive tasks regulated by the prefrontal cortex could also be impaired after exposure to mission relevant HZE-particle doses, which may prevent astronauts from performing complex executive functions. To assess the effects of mission relevant (20 cGy) doses of 1 GeV/u (56)Fe particles on executive function, male Wistar rats received either sham treatment or were irradiated and tested 3 months later for their ability to perform attentional set shifting. Compared to the controls, rats that received 20 cGy of 1 GeV/u (56)Fe particles showed significant impairments in their ability to complete the attentional set-shifting test, with only 17% of irradiated rats completing all stages as opposed to 78% of the control rats. The majority of failures (60%) occurred at the first reversal stage, and half of the remaining animals failed at the extra-dimensional shift phase of the studies. The irradiated rats that managed to complete the tasks did so with approximately the same ease as did the control rats. These observations suggest that exposure to mission relevant doses of 1 GeV/u (56)Fe particles results in the loss of functionality in several regions of the cortex: medical prefrontal cortex, anterior cingulated cortex, posterior cingulated cortex and the basal forebrain. Our observation that 20 cGy of 1 GeV/u (56)Fe particles is sufficient to impair the ability of rats to conduct attentional set-shifting raises the possibility that astronauts on prolonged deep space exploratory missions could subsequently develop deficits in executive function.

Low (20 cGy) doses of 1 GeV/u (56)Fe--particle radiation lead to a persistent reduction in the spatial learning ability of rats.

Exposure to galactic cosmic radiation (GCR) is considered to be a potential health risk in long-term space travel, and it represents a significant risk to the central nervous system (CNS). The most harmful component of GCR is the HZE [high-mass, highly charged (Z), high-energy] particles, e.g. (56)Fe. In previous ground-based experiments, exposure to high doses of HZE-particle radiation induced pronounced deficits in hippocampus-dependent learning and memory in rodents. Recent data suggest that glutamatergic transmission in hippocampal synaptosomes is impaired after low (60 cGy) doses of 1 GeV/u (56)Fe particles, which could lead to impairment of hippocampus-dependent spatial memory. To assess the effects of mission-relevant (20-60 cGy) doses of 1 GeV/u (56)Fe particles on hippocampus-dependent spatial memory, male Wistar rats either received sham treatment or were irradiated and tested 3 months later in the Barnes maze test. Compared to the controls, rats that received 20, 40 and 60 cGy 1 GeV/u (56)Fe particles showed significant impairments in their ability to locate the escape box in the Barnes maze, which was manifested by progressively increasing escape latency times over the 3 days of testing. However, this increase was not due to a lack of motivation of the rats to escape, because the total number of head pokes (and especially incorrect head pokes) remained constant over the test period. Given that rats exposed to X rays did not exhibit spatial memory impairments until >10 Gy was delivered, the RBE for 1 GeV/u (56)Fe-particle-induced hippocampal spatial memory impairment is ∼50. These data demonstrate that mission-relevant doses of 1 GeV/u (56)Fe particles can result in severe deficits in hippocampus-dependent neurocognitive tasks, and the extreme sensitivity of these processes to 1 GeV/u (56)Fe particles must arise due to the perturbation of multiple processes in addition to killing neuronal cells.

Low (60 cGy) doses of (56)Fe HZE-particle radiation lead to a persistent reduction in the glutamatergic readily releasable pool in rat hippocampal synaptosomes.

Exposure to galactic cosmic radiation (GCR) is considered to be a potential health risk in long-term space travel, and it represents a significant risk to the central nervous system (CNS). The most harmful component of GCR is the HZE [high-mass, highly charged (Z), high-energy] particles, e.g. (56)Fe. In ground-based experiments, exposure to HZE-particle radiation induces pronounced deficits in hippocampus-dependent learning and memory in rodents. The mechanisms underlying these impairments are mostly unknown, but some studies suggest that HZE-particle exposure perturbs the regulation of long-term potentiation (LTP) at the CA1 synapse in the hippocampus. In this study, we irradiated rats with 60 cGy of 1 GeV (56)Fe-particle radiation and established its impact on hippocampal glutamatergic neurotransmissions at 3 and 6 months after exposure. Exposure to 60 cGy (56)Fe-particle radiation significantly (P < 0.05) reduced hyperosmotic sucrose evoked [(3)H]-glutamate release from hippocampal synaptosomes, a measure of the readily releasable vesicular pool (RRP). This HZE-particle-induced reduction in the glutamatergic RRP persisted for at least 6 months after exposure. At 90 days postirradiation, there was a significant reduction in the expression of the NR1, NR2A and NR2B subunits of the glutamatergic NMDA receptor. The level of the NR2A protein remained suppressed at 180 days postirradiation, but the level of NR2B and NR1 proteins returned to or exceeded normal levels, respectively. Overall, this study shows that hippocampal glutamatergic transmission is sensitive to relative low doses of (56)Fe particles. Whether the observed HZE-particle-induced change in glutamate transmission, which plays a critical role in learning and memory, is the cause of HZE-particle-induced neurocognitive impairment requires further investigation.

The identification of serum biomarkers of high-let radiation exposure and biological sequelae.

In the event of a nuclear detonation, thousands of people will be exposed to non-lethal radiation doses. There are multiple long-term health concerns for exposed individuals who receive non-lethal radiation exposures. Low doses of radiation, especially of high linear energy transfer (LET) radiation, can lead to the development of neurocognitive defects. The identification of serum biomarkers that can be used to monitor the emergence of the long-term biological sequelae of radiation exposure, such as neurocognitive defects, would greatly help the post-exposure health monitoring of the affected population. The authors have determined the impact that cranial irradiation with 2 Gy of high LET (150 keV um) has on the ability of rats to perform spatial memory tasks, and identified serum protein changes that are biomarkers of radiation exposure and of radiation-induced neurocognitive impairment. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectroscopy (MALDI TOF-TOF) analysis of weak cation exchange (WCX) enriched serum protein preparations identified 23 proteins of interest: 10 were biomarkers of physical radiation dose, with six showing increased expression and four being undetectable in the irradiated rat serum. Four proteins were uniquely expressed in those rats that had good spatial memory and nine proteins were markers of bad spatial memory. This study provides proof of the concept that serum protein profiling can be used to identify biomarkers of radiation exposure and the emergence of radiation-sequelae in this rat model, and this approach could be easily applied to other systems to identify radiation biomarkers.

Synapsin II and calcium regulate vesicle docking and the cross-talk between vesicle pools at the mouse motor terminals.

The synapsins, an abundant and highly conserved family of proteins that associate with synaptic vesicles, have been implicated in regulating the synaptic vesicle cycle. However, it has not been determined whether synapsin directly regulates the number of docked vesicles. Here we document that reducing Ca(2+) concentration [Ca(2+)](o) in the extracellular medium from 2 to 0.5 mm led to an approximately 40% decrease in both docked and undocked synaptic vesicles in wild-type nerve terminals of the mouse diaphragm. The same treatment reduced the number of undocked vesicles in nerve terminals derived from synapsin II gene deleted animals, but surprisingly it did not decrease vesicle docking, indicating that synapsin II inhibits docking of synaptic vesicles at reduced [Ca(2+)](o). In accordance with the morphological findings, at reduced [Ca(2+)](o) synapsin II (-) terminals had a higher rate of quantal neurotransmitter release. Microinjection of a recombinant synapsin II protein into synapsin II (-) terminals reduced vesicular docking and inhibited quantal release, indicating a direct and selective synapsin II effect for regulating vesicle docking and, in turn, quantal release. To understand why [Ca(2+)](o) has a prominent effect on synapsin function, we investigated the effect of [Ca(2+)](o) on the distribution of synaptic vesicles and on the concentration of intraterminal Ca(2+). We found that reduced [Ca(2+)](o) conditions produce a decrease in intracellular Ca(2+) and overall vesicle depletion. To explore why at these conditions the role of synapsin II in vesicle docking becomes more prominent, we developed a quantitative model of the vesicle cycle, with a two step synapsin action in stabilizing the vesicle store and regulating vesicle docking. The results of the modelling were in a good agreement with the observed dependence of vesicle distribution on synapsin II and calcium deficiency.

The role of RIM1alpha in BDNF-enhanced glutamate release.

Brain-derived neurotrophic factor (BDNF) is known to activate proline-directed Ser/Thr protein kinases and to enhance glutamatergic transmission via a Rab3a-dependent molecular pathway. The identity of molecular targets in BDNF's action on Rab3a pathway, a synaptic vesicle protein involved in vesicle trafficking and synaptic plasticity, is not fully known. Here we demonstrate that BDNF enhances depolarization-evoked efflux of [(3)H]-glutamate from nerve terminals isolated from the CA1 region of the hippocampus. BDNF also potentiated hyperosmotic shock-evoked [(3)H]-glutamate efflux, indicating an effect on the size of the readily releasable pool. This effect of BDNF was completely abolished in nerve terminals derived from Rim1alphaKO (Rab3 interacting molecule 1alpha null mutant) mice. Using in vitro phosphorylation assays we identified two novel phosphorylation sites, Ser447 and Ser745 that were substrates for ERK2, a proline-directed kinase known to be activated by BDNF. The pSer447 site was phosphorylated under resting conditions in hippocampal CA1 nerve terminals and its phosphorylation was enhanced by BDNF treatment, as indicated by the use of a pSer447-RIM1alpha antibody we have developed. Together these findings identify RIM1alpha, a component of the Rab3a molecular pathway in mediating presynaptic plasticity, as a necessary factor in BDNF's enhancement of [(3)H]-glutamate efflux from hippocampal CA1 nerve terminals and indicate a possible role for RIM1alpha phosphorylation in BDNF-dependent presynaptic plasticity.

Transient fear-induced alterations in evoked release of norepinephrine and GABA in amygdala slices.

Presentation of a tonal cue that previously had been associated with a fearful experience (footshock) produces alterations in arousal and sleep that occur after the fearful cue is no longer presented. To begin investigating neurochemical mechanisms that may underlie the effects of fearful cue presentation, we measured release of [(3)H]-norepinephrine ([(3)H]-NE]) and [(14)C]-gamma-amino-butyric acid ([(14)C]-GABA) from brain regions known to regulate arousal states and REM sleep. Depolarization-evoked release of [(3)H]-NE from amygdalar slices of mice, which were trained to recognize a tone as a fearful cue, was suppressed at 2-3 h after exposure of animals to the fearful cue, but recovered after 4-5 h. Interestingly, depolarization-evoked release of [(14)C]-GABA was significantly increased in the amygdala, and also showed a tendency for enhancement in the hippocampus, NPO, and DRN at 2-3 h after cue presentation. The changes in [(14)C]-GABA release were also transient; 4-5 h after cue presentation no significant differences were detected between samples derived from experimental groups which experienced fearful or neutral cues. The similar time course of fearful cue-induced changes in neurotransmitter release and changes in arousal and REM sleep suggests that alterations in amygdalar neurotransmission may be involved in the changes in arousal and sleep that occur after fear.

Deletion of synapsins I and II genes alters the size of vesicular pools and rabphilin phosphorylation.

Previous studies established that genetic deletion of synapsins, synaptic vesicle-associated phosphoproteins that regulate neurotransmitter release, decreases the number of synaptic vesicles in nerve terminals. To investigate whether these changes affect the release properties of the remaining synaptic vesicles, we used a radioactive labeling technique to measure release independently of the total number of synaptic vesicles. 3H-glutamate and 14C-gamma-amino-butyric-acid (GABA) release from isolated nerve terminals prepared from the neocortex of synapsins I and II double knock-out mice (DKO) was assayed and compared to wild-type preparations. Hyperosmotic shock-evoked 3H-glutamate was reduced by 20+/-3% from DKO nerve terminals and potassium depolarization-evoked glutamate release was also decreased by 28+/-2%. Surprisingly, sucrose or potassium depolarization-evoked release of 14C-GABA was increased by 32+/-4% and 29+/-5%, respectively. The basal efflux of both 3H-glutamate and 14C-GABA increased by 17+/-2% and 12+/-2% from DKO nerve terminals. As lack of synapsins I and II, major phosphoproteins of synaptic vesicles, may lead to deregulation of phosphorylation events, we compared phosphorylation state of another synaptic vesicle protein, rabphilin. In DKO nerve terminals, membrane-associated rabphilin level was reduced by approximately 0.28-fold, its phosphorylation at 234serine was increased by approximately 1.61-fold whereas cytosolic rabphilin levels showed both more dramatic reduction in abundance, approximately 16.5-fold, and increase in phosphorylation, approximately 4.8-fold. Collectively, these data suggest that deletion of major synapsin isoforms leads to (1) deregulation of basal neurotransmission causing "leaky" basal release, (2) changes in either the size or mobilization of releasable or reserve pools, and (3) a decrease in rabphilin abundance accompanied by an increase in basal phosphorylation of the remaining rabphilin.

Adapter protein 14-3-3 is required for a presynaptic form of LTP in the cerebellum.

Long-term potentiation (LTP) of granule cell-Purkinje cell synapses in the mouse cerebellum requires phosphorylation by protein kinase A of the active-zone protein RIM1alpha at Ser413. Here, we show that the adapter protein 14-3-3 readily binds phosphorylated Ser413 in RIM1alpha, and that presynaptic transfection with a dominant-negative 14-3-3eta mutant, or a RIM1alpha mutant with enhanced 14-3-3 binding, inhibits LTP. Thus, RIM1alpha phosphorylation triggers presynaptic LTP in part through recruitment of 14-3-3 to phospho-Ser413-RIM1alpha.

Phosphorylation of RIM1alpha by PKA triggers presynaptic long-term potentiation at cerebellar parallel fiber synapses.

Presynaptic activation of protein kinase A (PKA) induces LTP in cerebellar parallel fiber synapses. Presynaptic LTP is known to require the active zone protein RIM1alpha, but the underlying induction mechanism remains unclear. We now show that PKA directly phosphorylates RIM1alpha at two sites. Using paired recordings from cultured cerebellar granule and Purkinje neurons, we demonstrate that LTP is absent in neurons from RIM1alpha KO mice but is rescued by presynaptic expression of RIM1alpha. Mutant RIM1alpha lacking the N-terminal phosphorylation site is unable to rescue LTP in RIM1alpha knockout neurons but selectively suppresses LTP in wild-type neurons. Our findings suggest that PKA-mediated phosphorylation of the active zone protein RIM1alpha at a single N-terminal site induces presynaptic LTP.

RIM1: an edge for presynaptic plasticity.

Pioneering work suggests that a synaptic active zone protein, RIM1, regulates both short- and long-term glutamatergic presynaptic plasticity at certain synapses. In short-term plasticity, RIM1 accelerates the priming of synaptic vesicles for fusion; by contrast, in long-term potentiation of mossy fiber synapses in the hippocampal CA3 region, phosphorylated RIM1 acts through an unknown molecular pathway to enhance release of the excitatory neurotransmitter glutamate.