Skewed distribution of spines is independent of presynaptic transmitter release and synaptic plasticity, and emerges early during adult neurogenesis.

Dendritic spines are crucial for excitatory synaptic transmission as the size of a spine head correlates with the strength of its synapse. The distribution of spine head sizes follows a lognormal-like distribution with more small spines than large ones. We analysed the impact of synaptic activity and plasticity on the spine size distribution in adult-born hippocampal granule cells from rats with induced homo- and heterosynaptic long-term plasticity in vivo and CA1 pyramidal cells from Munc13-1/Munc13-2 knockout mice with completely blocked synaptic transmission. Neither the induction of extrinsic synaptic plasticity nor the blockage of presynaptic activity degrades the lognormal-like distribution but changes its mean, variance and skewness. The skewed distribution develops early in the life of the neuron. Our findings and their computational modelling support the idea that intrinsic synaptic plasticity is sufficient for the generation, while a combination of intrinsic and extrinsic synaptic plasticity maintains lognormal-like distribution of spines.

Formation and Maintenance of Functional Spines in the Absence of Presynaptic Glutamate Release.

Dendritic spines are the major transmitter reception compartments of glutamatergic synapses in most principal neurons of the mammalian brain and play a key role in the function of nerve cell circuits. The formation of functional spine synapses is thought to be critically dependent on presynaptic glutamatergic signaling. By analyzing CA1 pyramidal neurons in mutant hippocampal slice cultures that are essentially devoid of presynaptic transmitter release, we demonstrate that the formation and maintenance of dendrites and functional spines are independent of synaptic glutamate release.

Transient oxytocin signaling primes the development and function of excitatory hippocampal neurons.

Beyond its role in parturition and lactation, oxytocin influences higher brain processes that control social behavior of mammals, and perturbed oxytocin signaling has been linked to the pathogenesis of several psychiatric disorders. However, it is still largely unknown how oxytocin exactly regulates neuronal function. We show that early, transient oxytocin exposure in vitro inhibits the development of hippocampal glutamatergic neurons, leading to reduced dendrite complexity, synapse density, and excitatory transmission, while sparing GABAergic neurons. Conversely, genetic elimination of oxytocin receptors increases the expression of protein components of excitatory synapses and excitatory synaptic transmission in vitro. In vivo, oxytocin-receptor-deficient hippocampal pyramidal neurons develop more complex dendrites, which leads to increased spine number and reduced γ-oscillations. These results indicate that oxytocin controls the development of hippocampal excitatory neurons and contributes to the maintenance of a physiological excitation/inhibition balance, whose disruption can cause neurobehavioral disturbances.

Automated and quantitative image analysis of ischemic dendritic blebbing using in vivo 2-photon microscopy data.

Ischemia induces a 'blebbing' of dendrites, a structural alteration where dendrites take on a 'beads on a string' appearance. We developed a toolkit program, BlebQuant, for quantitative automated bleb analysis to chart the morphology of dendrites labeled with GFP/YFP under normal conditions and after ischemia-induced damage. In vivo 2-photon data from mouse layer 5 neurons with apical dendritic tufts extending to the cortical surface were examined before, during, and after global ischemia. To quantify changes in dendritic structure, we used morphometric tools that exploit characteristic features of blebbing, distinguished as localized regions of spherical or ellipsoid swellings. By comparing acquired images during ischemia and reperfusion to a pre-ischemia reference image, our automated approach detected blebs based on defined eccentricity and area thresholds and quantified the percentage of blebbed dendrites based on a block-selection method. Our results indicate that the automated morphometric indices we employ yield results that correlate with manual assessment. The automated approach permits rapid and effective analysis of dendritic structure and may facilitate the study of ischemic dendritic damage.

In vivo 2-photon imaging of fine structure in the rodent brain: before, during, and after stroke.

The recent application of 2-photon microscopy to biological specimens has allowed investigators to examine individual synapses within live animals. The gain in resolution over conventional in vivo imaging techniques has been several orders of magnitude. We outline steps for the preparation and maintenance of animals for 2-photon microscopy of fine brain structure. We discuss the in vivo resolution of the method and the ability to image blood flow and synaptic structure in vivo. Applications of in vivo 2-photon microscopy include the study of synapse turnover in adult animals under normal conditions and during pathology such as stroke. In the case of stroke, 2-photon imaging has revealed marked swelling of dendrites and loss of spines within minutes of ischemic onset. Surprisingly, restoration of blood flow during reperfusion was associated with a return of relatively normal structure. Over longer time scales, 2-photon imaging revealed elevated rates of synaptogenesis within peri-infarct tissues recovering from stroke. These results provide an example of how high-resolution in vivo microscopy can be used to provide insight into both the acute pathology and recovery from stroke damage.

Imaging rapid redistribution of sensory-evoked depolarization through existing cortical pathways after targeted stroke in mice.

Evidence suggests that recovery from stroke damage results from the production of new synaptic pathways within surviving brain regions over weeks. To address whether brain function might redistribute more rapidly through preexisting pathways, we examined patterns of sensory-evoked depolarization in mouse somatosensory cortex within hours after targeted stroke to a subset of the forelimb sensory map. Brain activity was mapped with voltage-sensitive dye imaging allowing millisecond time resolution over 9 mm(2) of brain. Before targeted stroke, we report rapid activation of the forelimb area within 10 ms of contralateral forelimb stimulation and more delayed activation of related areas of cortex such as the hindlimb sensory and motor cortices. After stroke to a subset of the forelimb somatosensory cortex map, function was lost in ischemic areas within the forelimb map center, but maintained in regions 200-500 microm blood flow deficits indicating the size of a perfused, but nonfunctional, penumbra. In many cases, stroke led to only partial loss of the forelimb map, indicating that a subset of a somatosensory domain can function on its own. Within the forelimb map spared by stroke, forelimb-stimulated responses became delayed in kinetics, and their center of activity shifted into adjacent hindlimb and posterior-lateral sensory areas. We conclude that the focus of forelimb-specific somatosensory cortex activity can be rapidly redistributed after ischemic damage. Given that redistribution occurs within an hour, the effect is likely to involve surviving accessory pathways and could potentially contribute to rapid behavioral compensation or direct future circuit rewiring.

Simple and cost-effective hardware and software for functional brain mapping using intrinsic optical signal imaging.

We describe a simple and low-cost system for intrinsic optical signal (IOS) imaging using stable LED light sources, basic microscopes, and commonly available CCD cameras. IOS imaging measures activity-dependent changes in the light reflectance of brain tissue, and can be performed with a minimum of specialized equipment. Our system uses LED ring lights that can be mounted on standard microscope objectives or video lenses to provide a homogeneous and stable light source, with less than 0.003% fluctuation across images averaged from 40 trials. We describe the equipment and surgical techniques necessary for both acute and chronic mouse preparations, and provide software that can create maps of sensory representations from images captured by inexpensive 8-bit cameras or by 12-bit cameras. The IOS imaging system can be adapted to commercial upright microscopes or custom macroscopes, eliminating the need for dedicated equipment or complex optical paths. This method can be combined with parallel high resolution imaging techniques such as two-photon microscopy.

A novel method for processing resin-embedded specimens with metal implants for immunohistochemical labelling.

A major technical problem in the processing of resin-embedded tissues is the adhesion of the tissue sample on glass slides for immunohistochemical labelling. We therefore established a novel protocol for processing such specimens with improved attachment of the tissue sample during resin removal (deplastification). In order to demonstrate the feasibility of the procedure we employed a panel of smooth muscle cell maturation markers. The technique makes use of a silicone glue (Elastosil E41; Wacker Chemie, München, Germany) to attach the tissue samples to the glass slides. This allows resin dissolution in xylene/2-methoxyethylacetate without detachment of the sample from the slide. Our results demonstrate successful immunohistochemical labelling with primary antibodies directed against: smooth muscle actin, smooth muscle myosin, h-caldesmon, desmin, vimentin and von Willebrand factor. In conclusion, we have established a new and successful method for resin-embedded sample adhesion on glass slides. The developed protocol is feasible for investigation of cells which are involved in intimal proliferation following stent implantation.

Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope.

Investigators have begun to probe the role of individual surface arterioles in maintaining both the structure and function of cortical regions using vessel-specific clotting by in vivo photothrombosis after craniotomy in mice. To induce targeted strokes we describe a simple adaptation of a commercial upright Olympus BX51WI microscope, permitting light from a 532 nm laser to be directed into the back aperture of a high numerical aperture fluorescence objective. The system involves using a filter slot available on an Olympus BX series microscope to direct a collimated laser beam through the normal epifluorescence path to the objective back aperture resulting in focused photoactivation, with lateral and axial dimensions less than 3 and 5 microm, respectively. Existing fluorescence filters and dichroic mirrors are employed permitting one to safely target the green laser beam and view the clotting process based as red epifluorescence, either through the eye pieces or using a CCD camera. Interruption in blood flow can be confirmed using laser speckle microscopy. The positioning of the photothrombotic laser in this manner does not impede subsequent analysis of brain microcirculation using two-photon microscopy or other imaging methods. It is conceivable that this modification and laser system can also be used for other scenarios where targeted photoactivation or photobleaching would be required.

CAPS-1 and CAPS-2 are essential synaptic vesicle priming proteins.

Before transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca(2+) levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca(2+) triggered release of transmitters.

Functional inactivation of a fraction of excitatory synapses in mice deficient for the active zone protein bassoon.

Mutant mice lacking the central region of the presynaptic active zone protein Bassoon were generated to establish the role of this protein in the assembly and function of active zones as sites of synaptic vesicle docking and fusion. Our data show that the loss of Bassoon causes a reduction in normal synaptic transmission, which can be attributed to the inactivation of a significant fraction of glutamatergic synapses. At these synapses, vesicles are clustered and docked in normal numbers but are unable to fuse. Phenotypically, the loss of Bassoon causes spontaneous epileptic seizures. These data show that Bassoon is not essential for synapse formation but plays an essential role in the regulated neurotransmitter release from a subset of glutamatergic synapses.

Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming.

Synaptic vesicles must be primed to fusion competence before they can fuse with the plasma membrane in response to increased intracellular Ca2+ levels. The presynaptic active zone protein Munc13-1 is essential for priming of glutamatergic synaptic vesicles in hippocampal neurons. However, a small subpopulation of synapses in any given glutamatergic nerve cell as well as all gamma-aminobutyratergic (GABAergic) synapses are largely independent of Munc13-1. We show here that Munc13-2, the only Munc13 isoform coexpressed with Munc13-1 in hippocampus, is responsible for vesicle priming in Munc13-1 independent hippocampal synapses. Neurons lacking both Munc13-1 and Munc13-2 show neither evoked nor spontaneous release events, yet form normal numbers of synapses with typical ultrastructural features. Thus, the two Munc13 isoforms are completely redundant in GABAergic cells whereas glutamatergic neurons form two types of synapses, one of which is solely Munc13-1 dependent and lacks Munc13-2 whereas the other type employs Munc13-2 as priming factor. We conclude that Munc13-mediated vesicle priming is not a transmitter specific phenomenon but rather a general and essential feature of multiple fast neurotransmitter systems, and that synaptogenesis during development is not dependent on synaptic secretory activity.

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms.

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.