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Supplementary Figure 2 from Caffeine-Mediated Inhibition of Calcium Release Channel Inositol 1,4,5-Trisphosphate Receptor Subtype 3 Blocks Glioblastoma Invasion and Extends Survival

Supplementary Figure 1 from Caffeine-Mediated Inhibition of Calcium Release Channel Inositol 1,4,5-Trisphosphate Receptor Subtype 3 Blocks Glioblastoma Invasion and Extends Survival

Supplementary Figure 4 from Caffeine-Mediated Inhibition of Calcium Release Channel Inositol 1,4,5-Trisphosphate Receptor Subtype 3 Blocks Glioblastoma Invasion and Extends Survival

Supplementary Methods from Caffeine-Mediated Inhibition of Calcium Release Channel Inositol 1,4,5-Trisphosphate Receptor Subtype 3 Blocks Glioblastoma Invasion and Extends Survival

Many studies have implicated hippocampal dysregulation in the pathophysiology of alcohol use disorder (AUD). However, over the past twenty years, a growing body of evidence has revealed distinct functional roles of the dorsal (dHC) and ventral (vHC) hippocampal subregions, with the dHC being primarily involved in spatial learning and memory and the vHC regulating anxiety-and depressive-like behaviors. Notably, to our knowledge, no rodent studies have examined the effects of chronic ethanol exposure on synaptic transmission along the dorsal/ventral axis. To that end, we examined the effects of the chronic intermittent ethanol vapor exposure (CIE) model of AUD on dHC and vHC synaptic excitability. Adult male Long-Evans rats were exposed to CIE or air for 10 days (12 hrs/day; targeting blood ethanol levels of 175-225 mg%) and recordings were made 24 hours into withdrawal. As expected, this protocol increased anxiety-like behaviors on the elevated plus-maze. Extracellular recordings revealed marked CIE-associated increases in synaptic excitation in the CA1 region that were exclusively restricted to the ventral domain of the hippocampus. Western blot analysis of synaptoneurosomal fractions revealed that the expression of two proteins that regulate synaptic strength, GluA2 and SK2, was dysregulated in the vHC, but not the dHC, following CIE. Together, these findings suggest that the ventral CA1 region may be particularly sensitive to the maladaptive effects of chronic ethanol exposure and provide new insight into some of the neural substrates that may contribute to the negative affective state that develops during withdrawal.HighlightsChronic intermittent ethanol exposure produces robust increases in anxiety-like behavior in male Long Evans rats.Chronic intermittent ethanol exposure increases synaptic excitability in the ventral, but not the dorsal, domain of the hippocampus.These changes in excitability are associated with alterations in synaptoneurosomal expression of small conductance calcium-activated potassium channels and the GluA2 AMPA receptor subunit that are also restricted to the ventral hippocampus.

Alcohol use disorder (AUD) and anxiety/stressor disorders frequently co-occur and this dual diagnosis represents a major health and economic problem worldwide. The basolateral amygdala (BLA) is a key brain region that is known to contribute to the etiology of both disorders. Although many studies have implicated BLA hyperexcitability in the pathogenesis of AUD and comorbid conditions, relatively little is known about the specific efferent projections from this brain region that contribute to these disorders. Recent optogenetic studies have shown that the BLA sends a strong monosynaptic excitatory projection to the ventral hippocampus (vHC) and that this circuit modulates anxiety- and fear-related behaviors. However, it is not known if this pathway influences alcohol drinking-related behaviors. Here, we employed a rodent operant drinking regimen that procedurally separates appetitive (e.g. seeking) and consummatory (e.g. intake) behaviors, chemogenetics, and brain region-specific microinjections, to determine if BLA-vHC circuitry influences alcohol drinking-related measures. We first confirmed prior optogenetic findings that silencing this circuit reduced anxiety-like behaviors on the elevated plus-maze. We then demonstrated that inhibiting the BLA-vHC pathway significantly reduced appetitive alcohol drinking-related behaviors while having no effect on consummatory measures. Sucrose seeking measures were also reduced following chemogenetic inhibition of this circuit. Taken together, these findings provide the first indication that the BLA-vHC circuit may regulate appetitive alcohol drinking-related behaviors and add to a growing body of evidence suggesting that dysregulation of this pathway may contribute to the pathophysiology of AUD and anxiety/stressor-related disorders.HIGHLIGHTSThe basolateral amygdala sends a monosynaptic glutamatergic projection to the ventral hippocampusInhibiting this circuit reduces anxiety-like behaviors in male Long Evans ratsInhibition of this pathway also decreases operant alcohol seeking-related behaviors

Background Adenosine serves many functions within the CNS, including inhibitory and excitatory control of neurotransmission. The understanding of adenosine dynamics in the brain is of fundamental importance. The goal of the present study was to explore subsecond adenosine fluctuations in the rat brain in vivo . Method Long Evans rats were anesthetized and a carbon fiber electrode was positioned in the motor cortex or dorsal striatum. Real time electrochemical recordings were made at the carbon fiber electrodes every 100 ms by applying a triangular waveform (−0.4 to +1.5 V, 400 V/s). Adenosine spikes were identified by the background-subtracted cyclic voltammogram. Results The frequency of detected adenosine spikes was relatively stable in both tested regions, and the time intervals between spikes were regular and lasted from 1 to 5 s within an animal. Spike frequency ranged from 0.5 to 1.5 Hz in both the motor cortex and the dorsal striatum. Average spike amplitudes were 85 ± 11 and 66 ± 7 nM for the motor cortex and the dorsal striatum, respectively. Comparison with existing methods The current study established that adenosine signaling can operate on a fast time scale (within seconds) to modulate brain functions. Conclusions This finding suggests that spontaneous adenosine release may play a fast, dynamic role in regulating an organism's response to external events. Therefore, adenosine transmission in the brain may have characteristics similar to those of classical neurotransmitters, such as dopamine and norepinephrine.

It has long been appreciated that adolescence represents a uniquely vulnerable period when chronic exposure to stressors can precipitate the onset of a broad spectrum of psychiatric disorders and addiction in adulthood. However, the neurobiological substrates and the full repertoire of adaptations within these substrates making adolescence a particularly susceptible developmental stage are not well understood. Prior work has demonstrated that a rodent model of adolescent social isolation (aSI) produces robust and persistent increases in phenotypes relevant to anxiety/stressor disorders and alcohol addiction, including anxiogenesis, deficits in fear extinction, and increased ethanol consumption. Here, we used extracellular field recordings in hippocampal slices to investigate adaptations in synaptic function and synaptic plasticity arising from aSI. We demonstrate that this early life stressor leads to enhanced excitatory synaptic transmission and decreased levels of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Further, these changes were largely confined to the ventral hippocampus. As the ventral hippocampus is integral to neurocircuitry that mediates emotional behaviors, our results add to mounting evidence that aSI has profound effects on brain areas that regulate affective states. These studies also lend additional support to our recent proposal of the aSI model as a valid model of alcohol addiction vulnerability.

Protease-activated receptor-1 (PAR1) is an unusual G-protein coupled receptor (GPCR) that is activated through proteolytic cleavage by extracellular serine proteases. Although previous work has shown that inhibiting PAR1 activation is neuroprotective in models of ischemia, traumatic injury, and neurotoxicity, surprisingly little is known about PAR1's contribution to normal brain function. Here, we used PAR1-/- mice to investigate the contribution of PAR1 function to memory formation and synaptic function. We demonstrate that PAR1-/- mice have deficits in hippocampus-dependent memory. We also show that while PAR1-/- mice have normal baseline synaptic transmission at Schaffer collateral-CA1 synapses, they exhibit severe deficits in N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP). Mounting evidence indicates that activation of PAR1 leads to potentiation of NMDAR-mediated responses in CA1 pyramidal cells. Taken together, this evidence and our data suggest an important role for PAR1 function in NMDAR-dependent processes subserving memory formation and synaptic plasticity.

We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC50 values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo. Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of pKa values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (−)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.

Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation.

Ca2+ signaling is an important determining factor in many cellular processes, especially in cancer cell proliferation, motility and invasion. Glioblastoma is the deadliest brain cancer with its average survival time of less than a year, with the most prominent cellular feature being the ability of these cells to migrate to and invade the neighboring tissue. We hypothesized that disturbing the Ca2+ signaling pathway would decrease the propensity for these cells to migrate. Thus, we investigated the detailed Ca2+ signaling pathway of the glioblastoma cells in response to various receptor tyrosine kinases (RTK) and G-protein coupled receptor (GPCR) agonists. Here we report that caffeine, which is a well-known activator of ryanodine receptors (RyRs), paradoxically inhibits inositol-1, 4, 5-triphospate receptor(IP3R)-mediated Ca2+ increase by selectively targeting IP3R subtype 3(IP3R3), whose mRNA expression is significantly increased in glioblastoma cells. Consequently, by inhibiting IP3R3-mediated Ca2+ release, caffeine was found to inhibit the invasion and migration of various glioblastoma cell lines in scrape motility, Matrigel invasion, soft agar, and brain slice implantation assays. In a mouse xenograft model of glioblastoma, caffeine intake via drinking water greatly increased mean survival duration of subject animals. These findings propose IP3R3 as a novel target for glioblastoma treatment and that caffeine may be a useful adjunct therapy that slows glioblastoma invasion and migration by selectively targeting IP3R3.

The rodent pilocarpine model of epilepsy exhibits hippocampal sclerosis and spontaneous seizures and thus resembles human temporal lobe epilepsy. Use of the many available mouse mutants to study this epilepsy model would benefit from a detailed neuropathology study. To identify new features of epileptogenesis, we characterized glial and neuronal pathologies after pilocarpine-induced status epilepticus (SE) in CF1 and C57BL/6 mice focusing on the hippocampus. All CF1 mice showed spontaneous seizures by 17-27 days after SE. By 6 h there was virtually complete loss of hilar neurons, but the extent of pyramidal cell death varied considerably among mice. In the mossy fiber pathway, neuropeptide Y (NPY) was persistently upregulated beginning 1 day after SE; NPY immunoreactivity in the supragranular layer after 31 days indicated mossy fiber sprouting. beta2 microglobulin-positive activated microglia, normally absent in brains without SE, became abundant over 3-31 days in regions of neuronal loss, including the hippocampus and the amygdala. Astrogliosis developed after 10 days in damaged areas. Amyloid precursor protein immunoreactivity in the thalamus at 10 days suggested delayed axonal degeneration. The mortality after pilocarpine injection was very high in C57BL/6 mice from Jackson Laboratories but not those from Charles River, suggesting that mutant mice in the C57BL/6(JAX) strain will be difficult to study in the pilocarpine model, although their neuropathology was similar to CF1 mice. Major neuropathological changes not previously studied in the rodent pilocarpine model include widespread microglial activation, delayed thalamic axonal death, and persistent NPY upregulation in mossy fibers, together revealing extensive and persistent glial as well as neuronal pathology.

SummaryMutations that cause intellectual disability (ID) and autism spectrum disorder (ASD) are commonly found in genes that encode for synaptic proteins. However, it remains unclear how mutations that disrupt synapse function impact intellectual ability. In the SYNGAP1 mouse model of ID/ASD, we found that dendritic spine synapses develop prematurely during the early postnatal period. Premature spine maturation dramatically enhanced excitability in the developing hippocampus, which corresponded with the emergence of behavioral abnormalities. Inducing SYNGAP1 mutations after critical developmental windows closed had minimal impact on spine synapse function, whereas repairing these pathogenic mutations in adulthood did not improve behavior and cognition. These data demonstrate that SynGAP protein acts as a critical developmental repressor of neural excitability that promotes the development of life-long cognitive abilities. We propose that the pace of dendritic spine synapse maturation in early life is a critical determinant of normal intellectual development.