Differential Subcellular Distribution and Release Dynamics of Cotransmitted Cholinergic and GABAergic Synaptic Inputs Modify Dopaminergic Neuronal Excitability.

We identified three types of monosynaptic cholinergic inputs spatially arranged onto medial substantia nigra dopaminergic neurons in male and female mice: cotransmitted acetylcholine (ACh)/GABA, GABA-only, and ACh only. There was a predominant GABA-only conductance along lateral dendrites and soma-centered ACh/GABA cotransmission. In response to repeated stimulation, the GABA conductance found on lateral dendrites decremented less than the proximally located GABA conductance, and was more effective at inhibiting action potentials. While soma-localized ACh/GABA cotransmission showed depression of the GABA component with repeated stimulation, ACh-mediated nicotinic responses were largely maintained. We investigated whether this differential change in inhibitory/excitatory inputs leads to altered neuronal excitability. We found that a depolarizing current or glutamate preceded by cotransmitted ACh/GABA was more effective in eliciting an action potential compared with current, glutamate, or ACh/GABA alone. This enhanced excitability was abolished with nicotinic receptor inhibitors, and modulated by T- and L-type calcium channels, thus establishing that activity of multiple classes of ion channels integrates to shape neuronal excitability.SIGNIFICANCE STATEMENT Our laboratory has previously discovered a population of substantia nigra dopaminegic neurons (DA) that receive cotransmitted ACh and GABA. This study used subcellular optogenetic stimulation of cholinergic presynaptic terminals to map the functional ACh and GABA synaptic inputs across the somatodendritic extent of substantia nigra DA neurons. We determined spatially clustered GABA-only inputs on the lateral dendrites while cotransmitted ACh and GABA clustered close to the soma. We have shown that the action of GABA and ACh in cotransmission spatially clustered near the soma play a critical role in enhancing glutamate-mediated neuronal excitability through the activation of T- and L-type voltage-gated calcium channels.

Brain Ultrastructure: Putting the Pieces Together.

Unraveling the fine structure of the brain is important to provide a better understanding of its normal and abnormal functioning. Application of high-resolution electron microscopic techniques gives us an unprecedented opportunity to discern details of the brain parenchyma at nanoscale resolution, although identifying different cell types and their unique features in two-dimensional, or three-dimensional images, remains a challenge even to experts in the field. This article provides insights into how to identify the different cell types in the central nervous system, based on nuclear and cytoplasmic features, amongst other unique characteristics. From the basic distinction between neurons and their supporting cells, the glia, to differences in their subcellular compartments, organelles and their interactions, ultrastructural analyses can provide unique insights into the changes in brain function during aging and disease conditions, such as stroke, neurodegeneration, infection and trauma. Brain parenchyma is composed of a dense mixture of neuronal and glial cell bodies, together with their intertwined processes. Intracellular components that vary between cells, and can become altered with aging or disease, relate to the cytoplasmic and nucleoplasmic density, nuclear heterochromatin pattern, mitochondria, endoplasmic reticulum and Golgi complex, lysosomes, neurosecretory vesicles, and cytoskeletal elements (actin, intermediate filaments, and microtubules). Applying immunolabeling techniques to visualize membrane-bound or intracellular proteins in neurons and glial cells gives an even better appreciation of the subtle differences unique to these cells across contexts of health and disease. Together, our observations reveal how simple ultrastructural features can be used to identify specific changes in cell types, their health status, and functional relationships in the brain.

Acute Δ9-tetrahydrocannabinol prompts rapid changes in cannabinoid CB1 receptor immunolabeling and subcellular structure in CA1 hippocampus of young adult male mice.

The use and abuse of cannabis can be associated with significant pathophysiology, however, it remains unclear whether (1) acute administration of Δ-9-tetrahydrocannabinol (THC) during early adulthood alters the cannabinoid type 1 (CB1 ) receptor localization and expression in cells of the brain, and (2) THC produces structural brain changes. Here we use electron microscopy and a highly sensitive pre-embedding immunogold method to examine CB1 receptors in the hippocampus cornu ammonis subfield 1 (CA1) 30 min after male mice were exposed to a single THC injection (5 mg/kg). The findings show that acute exposure to THC can significantly decrease the percentage of CB1 receptor immunopositive terminals making symmetric synapses, mitochondria, and astrocytes. The percentage of CB1 receptor-labeled terminals forming asymmetric synapses was unaffected. Lastly, CB1 receptor expression was significantly lower at terminals of symmetric and asymmetric synapses as well as in mitochondria. Structurally, CA1 dendrites were significantly larger, and contained more spines and mitochondria following acute THC administration. The area of the dendritic spines, synaptic terminals, mitochondria, and astrocytes decreased significantly following acute THC exposure. Altogether, these results indicate that even a single THC exposure can have a significant impact on CB1 receptor expression, and can alter CA1 ultrastructure, within 30 min of drug exposure. These changes may contribute to the behavioral alterations experienced by young individuals shortly after cannabis intoxication.

Unlocking the brain: A new method for Western blot protein detection from fixed brain tissue.

BACKGROUND: Aldehyde fixation is a common process used to preserve the complex structure of biological samples ex vivo. This method of fixation relies on the formation of covalent bonds between aldehydes and amines present in the biomolecules of the sample. Aldehyde fixation is routinely performed in histological studies, however fixed tissue samples are rarely used for non-histological purposes as the fixation process is thought to make brain tissue unsuitable for traditional proteomic analyses such as Western blot. Advances in antigen-retrieval procedures have allowed detectable levels of protein to be solubilized from formaldehyde fixed tissue, opening the door for aldehyde-fixed samples to be used in both histological and proteomic approaches. NEW METHOD: Here, we developed a series of antigen-retrieval steps for use on fixed-brain lysates to make them suitable for analysis by Western blot. RESULTS: Prolonged exposure of the tissue homogenate to high temperature (90 °C for 2 h) in the presence of a concentrated formaldehyde scavenger and ionic detergent was sufficient to reveal a variety of synaptic and non-synaptic proteins on membrane blots. CONCLUSION: This protocol has significant utility for future studies using fixed tissue samples in a variety of neuropathological conditions.

Intermittent ethanol exposure during adolescence impairs cannabinoid type 1 receptor-dependent long-term depression and recognition memory in adult mice.

Binge drinking is a significant problem in adolescent populations, and because of the reciprocal interactions between ethanol (EtOH) consumption and the endocannabinoid (eCB) system, we sought to determine if adolescent EtOH intake altered the localization and function of the cannabinoid 1 (CB1) receptors in the adult brain. Adolescent mice were exposed to a 4-day-per week drinking in the dark (DID) procedure for a total of 4 weeks and then tested after a 2-week withdrawal period. Field excitatory postsynaptic potentials (fEPSPs), evoked by medial perforant path (MPP) stimulation in the dentate gyrus molecular layer (DGML), were significantly smaller. Furthermore, unlike control animals, CB1 receptor activation did not depress fEPSPs in the EtOH-exposed animals. We also examined a form of excitatory long-term depression that is dependent on CB1 receptors (eCB-eLTD) and found that it was completely lacking in the animals that consumed EtOH during adolescence. Histological analyses indicated that adolescent EtOH intake significantly reduced the CB1 receptor distribution and proportion of immunopositive excitatory synaptic terminals in the medial DGML. Furthermore, there was decreased binding of [35S]guanosine-5*-O-(3-thiotriphosphate) ([35S] GTPγS) and the guanine nucleotide-binding (G) protein Gαi2 subunit in the EtOH-exposed animals. Associated with this, there was a significant increase in monoacylglycerol lipase (MAGL) mRNA and protein in the hippocampus of EtOH-exposed animals. Conversely, deficits in eCB-eLTD and recognition memory could be rescued by inhibiting MAGL with JZL184. These findings indicate that repeated exposure to EtOH during adolescence leads to long-term deficits in CB1 receptor expression, eCB-eLTD, and reduced recognition memory, but that these functional deficits can be restored by treatments that increase endogenous 2-arachidonoylglycerol.

Time and exposure to serotonin affect releasability of recycled vesicles at crayfish claw opener muscle synapses.

The crayfish claw opener neuromuscular junction is a biological model for studying presynaptic neuromodulation by serotonin (5HT) and synaptic vesicle recycling. It has been hypothesized that 5HT enhances release by recruiting a population of either previously nonrecycling or "reluctant" vesicles to increase the readily releasable pool. To determine if 5HT activates a distinct population of synaptic vesicles, recycling membranes were labeled with the membrane dye, FM1-43. Unloading (destaining) protocols could not resolve a population of vesicles that were only releasable in the presence of 5HT. Instead, we conclude synaptic vesicles change behavior in axon terminals independent of 5HT, becoming less likely to exocytose and unload dye over periods of >1 hr after recycling. We hypothesized this to be due to the slow conversion of a portion of recycled vesicles to a difficult to release state. The possibility that vesicles in these pools were spatially separated within the terminal was tested using photoconversion of FM1-43 and transmission electron microscopy. The location of FM1-43-labeled vesicles fixed 2 min following 3 min of 20-Hz stimulation did not reveal preferential localization of recycling vesicles specifically near release sites and the distribution of labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 s following stimulation contained a significant proportion of vesicular structures equivalent in diameter to 2-5 regular vesicles, with multivesicular bodies and calveoli rarely seen, suggesting that endocytosis during sustained release at crayfish terminals occurs via multiple routes, most commonly through large "vesicle" intermediates.

Endocannabinoid long-term depression revealed at medial perforant path excitatory synapses in the dentate gyrus.

The endocannabinoid system modulates synaptic plasticity in the hippocampus, but a link between long-term synaptic plasticity and the type 1 cannabinoid (CB1) receptor at medial perforant path (MPP) synapses remains elusive. Here, immuno-electron microscopy in adult mice showed that ∼26% of the excitatory synaptic terminals in the middle 1/3 of the dentate molecular layer (DML) contained CB1 receptors, and field excitatory postsynaptic potentials evoked by MPP stimulation were inhibited by CB1 receptor activation. In addition, MPP stimulation at 10 Hz for 10 min triggered CB1 receptor-dependent excitatory long-term depression (eCB-eLTD) at MPP synapses of wild-type mice but not on CB1-knockout mice. This eCB-eLTD was group I mGluR-dependent, required intracellular calcium influx and 2-arachydonoyl-glycerol (2-AG) synthesis but did not depend on N-methyl-d-aspartate (NMDA) receptors. Overall, these results point to a functional role for CB1 receptors with eCB-eLTD at DML MPP synapses and further involve these receptors in memory processing within the adult brain.

High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain.

Activation of type 1 cannabinoid (CB1) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB1 receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB1 receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB1 receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (e.g., glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB1 receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB1 receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB1 receptors and protein markers of specific brain cells or synapses (e.g., GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB1 receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.

Ultrastructural analysis of blood-brain barrier breakdown in the peri-infarct zone in young adult and aged mice.

Following ischemia, the blood-brain barrier is compromised in the peri-infarct zone leading to secondary injury and dysfunction that can limit recovery. Currently, it is uncertain what structural changes could account for blood-brain barrier permeability, particularly with aging. Here we examined the ultrastructure of early and delayed changes (3 versus 72 h) to the blood-brain barrier in young adult and aged mice (3-4 versus 18 months) subjected to photothrombotic stroke. At both time points and ages, permeability was associated with a striking increase in endothelial caveolae and vacuoles. Tight junctions were generally intact although small spaces were detected in a few cases. In young mice, ischemia led to a significant increase in pericyte process area and vessel coverage whereas these changes were attenuated with aging. Stroke led to an expansion of the basement membrane region that peaked at 3 h and partially recovered by 72 h in both age groups. Astrocyte endfeet and their mitochondria were severely swollen at both times points and ages. Our results suggest that blood-brain barrier permeability in young and aged animals is mediated by transcellular pathways (caveolae/vacuoles), rather than tight junction loss. Further, our data indicate that the effects of ischemia on pericytes and basement membrane are affected by aging.

Delayed inhibition of VEGF signaling after stroke attenuates blood-brain barrier breakdown and improves functional recovery in a comorbidity-dependent manner.

Diabetes is a common comorbidity in stroke patients and a strong predictor of poor functional outcome. To provide a more mechanistic understanding of this clinically relevant problem, we focused on how diabetes affects blood-brain barrier (BBB) function after stroke. Because the BBB can be compromised for days after stroke and thus further exacerbate ischemic injury, manipulating its function presents a unique opportunity for enhancing stroke recovery long after the window for thrombolytics has passed. Using a mouse model of Type 1 diabetes, we discovered that ischemic stroke leads to an abnormal and persistent increase in vascular endothelial growth factor receptor 2 (VEGF-R2) expression in peri-infarct vascular networks. Correlating with this, BBB permeability was markedly increased in diabetic mice, which could not be prevented with insulin treatment after stroke. Imaging of capillary ultrastructure revealed that BBB permeability was associated with an increase in endothelial transcytosis rather than a loss of tight junctions. Pharmacological inhibition (initiated 2.5 d after stroke) or vascular-specific knockdown of VEGF-R2 after stroke attenuated BBB permeability, loss of synaptic structure in peri-infarct regions, and improved recovery of forepaw function. However, the beneficial effects of VEGF-R2 inhibition on stroke recovery were restricted to diabetic mice and appeared to worsen BBB permeability in nondiabetic mice. Collectively, these results suggest that aberrant VEGF signaling and BBB dysfunction after stroke plays a crucial role in limiting functional recovery in an experimental model of diabetes. Furthermore, our data highlight the need to develop more personalized stroke treatments for a heterogeneous clinical population.

α4* Nicotinic acetylcholine receptors modulate experience-based cortical depression in the adult mouse somatosensory cortex.

The molecular mechanisms that mediate experience-based changes in the function of the cerebral cortex, particularly in the adult animal, are poorly understood. Here we show using in vivo voltage-sensitive dye imaging, that whisker trimming leads to depression of whisker-evoked sensory responses in primary, secondary and associative somatosensory cortical regions. Given the importance of cholinergic neurotransmission in cognitive and sensory functions, we examined whether α4-containing (α4*) nicotinic acetylcholine receptors (nAChRs) mediate cortical depression. Using knock-in mice that express YFP-tagged α4 nAChRs subunits, we show that whisker trimming selectively increased the number α4*-YFP nAChRs in layer 4 of deprived barrel columns within 24 h, which persisted until whiskers regrew. Confocal and electron microscopy revealed that these receptors were preferentially increased on the cell bodies of GABAergic neurons. To directly link these receptors with functional cortical depression, we show that depression could be induced in normal mice by topical application or micro-injection of α4* nAChR agonist in the somatosensory cortex. Furthermore, cortical depression could be blocked after whisker trimming with chronic infusions of an α4* nAChR antagonist. Collectively, these results uncover a new role for α4* nAChRs in regulating rapid changes in the functional responsiveness of the adult somatosensory cortex.

Nap1-mediated actin remodeling is essential for mammalian myoblast fusion.

Myoblast fusion is crucial for the formation, growth, maintenance and regeneration of healthy skeletal muscle. Unfortunately, the molecular machinery, cell behaviors, and membrane and cytoskeletal remodeling events that govern fusion and myofiber formation remain poorly understood. Using time-lapse imaging approaches on mouse C2C12 myoblasts, we identify discrete and specific molecular events at myoblast membranes during fusion and myotube formation. These events include rearrangement of cell shape from fibroblast to spindle-like morphologies, changes in lamellipodial and filopodial extensions during different periods of differentiation, and changes in membrane alignment and organization during fusion. We find that actin-cytoskeleton remodeling is crucial for these events: pharmacological inhibition of F-actin polymerization leads to decreased lamellipodial and filopodial extensions and to reduced myoblast fusion. Additionally, shRNA-mediated inhibition of Nap1, a member of the WAVE actin-remodeling complex, results in accumulations of F-actin structures at the plasma membrane that are concomitant with a decrease in myoblast fusion. Our data highlight distinct and essential roles for actin cytoskeleton remodeling during mammalian myoblast fusion, provide a platform for cellular and molecular dissection of the fusion process, and suggest a functional conservation of Nap1-regulated actin-cytoskeleton remodeling during myoblast fusion between mammals and Drosophila.

Fabrication of metal nanoparticles using toroidal plasmid DNA as a sacrificial mold.

A new method for synthesizing gold, nickel, and cobalt metal nanoparticles at room temperature from metal salts employing plasmid DNA in a toroidal topology as a sacrificial mold is presented. The diameter of the toroidal DNA drives the formation and size of the nanoparticle, and UV light initiates the oxidation of the DNA and concomitant reduction of the DNA bound metal ions. The nanoparticles were characterized by atomic force microscopy (AFM), transmission electron microscopy (TEM), and electron diffraction (ED).

What the buzz was all about: superfast song muscles rattle the tymbals of male periodical cicadas.

Male cicadas produce mating calls by oscillating a pair of superfast tymbal muscles in their anterior abdominal cavity that pull on and buckle stiff-ribbed cuticular tymbal membranes located beneath the folded wings. The functional anatomy and rattling of the tymbal organ in 17 yr periodical cicada, Magicicada cassini (Brood X), were revealed by high-resolution microcomputed tomography, magnetic resonance imaging, electron microscopy, and laser vibrometry to understand the mechanism of sound production in these insects. Each 50 Hz muscle contraction yielded five to six stages of rib buckling in the tymbal, and a small release of muscle tension resulted in a rapid recovery due to the spring-loaded nature of the stiff ribs in the resilin-rich tymbal. The tymbal muscle sarcomeres have thick and thin filaments that are 30% shorter than those in flight muscles, with Z-bands that were thicker and configured into novel perforated hexagonal lattices. Caffeine-treated fibers supercontracted by allowing thick filaments to traverse the Z-band through its open lattice. This superfast sonic muscle illustrates design features, especially the matching hexagonal symmetry of the myofilaments and the perforated Z-band that contribute to high-speed contractions, long endurance, and potentially supercontraction needed for producing enduring mating songs and choruses.

Myosin flares and actin leptomeres as myofibril assembly/disassembly intermediates in sonic muscle fibers.

The sonic muscle of type 1 male midshipman fish produces loud and enduring mating calls. Each sonic muscle fiber contains a tubular contractile apparatus with radially arranged myofibrillar plates encased in a desmin-rich cytoskeleton that is anchored to broad Z bands (approximately 1.2 micro m wide). Immunomicroscopy has revealed patches of myosin-rich "flares" emanating from the contractile tubes into the peripheral sarcoplasm along the length of the fibers. These flares contain swirls of thick filaments devoid of associated thin filaments. In other regions of the sarcoplasm at the inner surface of the sarcolemma and near Z bands, abundant ladder-like leptomeres occur with rungs every 160 nm. Leptomeres consist of dense arrays of filaments (approximately 4 nm) with a structure that resembles myofibrillar Z band structure. We propose that flares and leptomeres are distinct filamentous arrays representing site-specific processing of myofibrillar components during the assembly and disassembly of the sarcomere. Recent reports that myosin assembles into filamentous aggregates before incorporating into the A band in the skeletal muscles of vertebrates and Caenorhabditis elegans suggest that sonic fibers utilize a similar pathway. Thus, sonic muscle fibers, with their tubular design and abundant sarcoplasmic space, may provide an attractive muscle model to identify myofibrillar intermediates by structural and molecular techniques.

Concentric intermediate filament lattice links to specialized Z-band junctional complexes in sonic muscle fibers of the type I male midshipman fish.

Type I male midshipman fish produce high-frequency hums for prolonged durations using sonic muscle fibers, each of which contains a hollow tube of radially oriented thin and flat myofibrils that display extraordinarily wide ( approximately 1.2 microm) Z bands. We have revealed an elaborate cytoskeletal network of desmin filaments associated with the contractile cylinder that form interconnected concentric ring structures in the core and periphery at the level of the Z bands. Stretch and release of single fibers revealed reversible length changes in the elastic desmin lattice. This lattice is linked to Z bands via novel intracellular desmosome-like junctional complexes that collectively form a ring, termed the "Z corset," around the periphery and within the core of the cylinder. The junctional complex consists of regularly spaced parallel approximately 900-nm-long cytoskeletal rods, or "Z bars," interconnected with slender (3-4 nm) plectin-positive filaments. Z bars are linked to the Z band by plectin filaments and on the opposite side to a dense mesh of desmin filaments. Adjacent Z bands are linked by slender filaments that appear to suspend sarcotubules. We propose that the highly reinforced elastic desmin cytoskeleton and the unique Z band junctions are structural adaptations that enable the muscles' high-frequency and high-endurance activity.

Evidence for an extracellular matrix bridge guiding proepicardial cell migration to the myocardium of chick embryos.

During heart development, the proepicardium (PE) gives rise to cells of the epicardial epithelium, connective tissue of the subepicardium and the myocardium, and smooth muscle, endothelium, and connective tissue of the coronary arteries. The PE arises as an outgrowth of the pericardial serosa at embryonic day 2 (Hamburger and Hamilton stage [HH] 14) of chick development. Between stages HH14 and HH17, multicellular villous projections extend from the PE toward the dorsal aspect of the lesser curvature of the myocardium. On reaching the atrioventricular (AV) junction, the cells spread over the myocardium, eventually enveloping the complete heart surface as a simple squamous epithelium. Although the lineage of the PE cells is well established, it remains uncertain how cells of the PE reach the myocardial surface and specifically target the AV junction. By using a combination of serial section reconstructions, immunofluorescence, and electron microscopy, we have identified an extracellular matrix bridge (ECMB) spanning the coelomic cavity between the PE and the myocardium. The ECMB is first detectable at HH14 and persists until the PE contacts the bare myocardial surface. This ECMB stains intensely with ruthenium red and Alcian blue, contains heparan sulfate and fibronectin, and exhibits both fibrillar and globular ultrastructure, reminiscent of proteoglycans. After PE attachment to the myocardium (HH16-HH17), the subepicardium exhibited strong staining for heparan sulfate. Heparinase injection into the pericardial coelom at HH15 resulted in aberrant development of the primordial epicardium. On the basis of these studies, we suggest that the ECMB may participate in migration and targeting of the PE to the myocardium.