Dendritic spines receive most synaptic inputs in the forebrain. Their morphology, with a spine head isolated from the dendrite by a slender neck, indicates a potential role in isolating inputs. Indeed, biochemical compartmentalization occurs at spine heads because of the diffusional bottleneck created by the spine neck. Here we investigate whether the spine neck also isolates inputs electrically. Using two-photon uncaging of glutamate on spine heads from mouse layer-5 neocortical pyramidal cells, we find that the amplitude of uncaging potentials at the soma is inversely proportional to neck length. This effect is strong and independent of the position of the spine in the dendritic tree and size of the spine head. Moreover, spines with long necks are electrically silent at the soma, although their heads are activated by the uncaging event, as determined with calcium imaging. Finally, second harmonic measurements of membrane potential reveal an attenuation of somatic voltages into the spine head, an attenuation directly proportional to neck length. We conclude that the spine neck plays an electrical role in the transmission of membrane potentials, isolating synapses electrically.second harmonic ͉ electrical isolation ͉ uncaging ͉ cortex ͉ glutamate T he dendritic spine is a ubiquitous feature in the nervous system, whose function is still poorly understood and heavily investigated (1). Spines are recipients of excitatory inputs in many neurons, including pyramidal cells (2), but excitatory inputs in nonspiny neurons contact dendritic shafts. Therefore, rather than just serving as recipients of inputs, spines likely perform a specific function with those inputs. Indeed, spines are calcium compartments and can therefore restrict local biochemical reactions to single inputs (3, 4). Nevertheless, nonspiny neurons can also perform similar calcium compartmentalization (5, 6), so it is conceivable that spines could implement an additional function.Theoretical work, spanning several decades, has proposed that spines could play an important role in altering synaptic potentials (7-12) (for a recent review, see ref. 13). Because of the resistance of the spine neck, spines could electrically isolate inputs and thus prevent input resistance variations in the dendrite during synaptic transmission (8). Thus, excitatory synaptic potentials could be filtered when they reach the dendrite (8-10, 12).The resistance of the spine neck, a crucial variable in ascertaining the electrical function of the spine, has never been measured. Estimates made from passive cable models (14) or diffusional coupling (15) would make its value too low to significantly filter synaptic potentials. At the same time, recent diffusional estimates indicate that neck resistances could be higher (16). Indeed, in our recent work examining input integration, we found that potentials onto spines sum linearly, whereas depolarizations on dendritic shafts shunt each other (R.A., K.B.E., and R.Y., unpublished work). Thus, our data would imply that spines isolate input...

Laser microscopy has generally poor temporal resolution, caused by the serial scanning of each pixel. This is a signifi cant problem for imaging or optically manipulating neural circuits, since neuronal activity is fast. To help surmount this limitation, we have developed a "scanless" microscope that does not contain mechanically moving parts. This microscope uses a diffractive spatial light modulator (SLM) to shape an incoming two-photon laser beam into any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision. To demonstrate the usefulness of this microscope, we perform two-photon uncaging of glutamate to activate dendritic spines and cortical neurons in brain slices. We also use it to carry out fast (60 Hz) two-photon calcium imaging of action potentials in neuronal populations. Thus, SLM microscopy appears to be a powerful tool for imaging and optically manipulating neurons and neuronal circuits. Moreover, the use of SLMs expands the fl exibility of laser microscopy, as it can substitute traditional simple fi xed lenses with any calculated lens function.

Most excitatory inputs in the mammalian brain are made on dendritic spines, rather than on dendritic shafts. Spines compartmentalize calcium, and this biochemical isolation can underlie input-specific synaptic plasticity, providing a raison d'etre for spines. However, recent results indicate that the spine can experience a membrane potential different from that in the parent dendrite, as though the spine neck electrically isolated the spine. Here we use two-photon calcium imaging of mouse neocortical pyramidal neurons to analyze the correlation between the morphologies of spines activated under minimal synaptic stimulation and the excitatory postsynaptic potentials they generate. We find that excitatory postsynaptic potential amplitudes are inversely correlated with spine neck lengths. Furthermore, a spike timing-dependent plasticity protocol, in which two-photon glutamate uncaging over a spine is paired with postsynaptic spikes, produces rapid shrinkage of the spine neck and concomitant increases in the amplitude of the evoked spine potentials. Using numerical simulations, we explore the parameter regimes for the spine neck resistance and synaptic conductance changes necessary to explain our observations. Our data, directly correlating synaptic and morphological plasticity, imply that long-necked spines have small or negligible somatic voltage contributions, but that, upon synaptic stimulation paired with postsynaptic activity, they can shorten their necks and increase synaptic efficacy, thus changing the input/output gain of pyramidal neurons.STDP | neocortex | basal dendrites D endritic spines are found in neurons throughout the central nervous system (1), and in pyramidal neurons receive the majority of excitatory inputs, whereas dendritic shafts are normally devoid of glutamatergic synapses (2-7). These facts suggest that spines are likely to play an essential role in neural circuits (1), although it is still unclear exactly what this role is (8, 9). Because of their peculiar morphology, hypotheses regarding the specific function of spines have focused on their role in biochemical compartmentalization, whereby a small spine head, where the excitatory synapse is located, is separated from the parent dendrite by a thin neck, isolating the spine cytoplasm from the dendrite (10). Indeed, spines are diffusionally restricted from dendrites (11-13) and compartmentalize calcium after synaptic stimulation (14-16). This local biochemistry and the high calcium accumulations observed following temporal pairing of neuronal input and output (14,17,18) are thought to be responsible for input-specific synaptic plasticity (19-21). However, besides this biochemical role, spines have also been hypothesized to play an electrical role, altering excitatory postsynaptic potentials (EPSPs) (22-30). Consistent with this idea, activating spines with two-photon uncaging of glutamate generates potentials whose amplitudes are inversely proportional to the length of the spine neck (31), and these responses are much larger in spines than...

We describe neurobiological applications of RuBi-Glutamate, a novel caged-glutamate compound based on ruthenium photochemistry. RuBi-Glutamate can be excited with visible wavelengths and releases glutamate after one-or two-photon excitation. It has high quantum effi ciency and can be used at low concentrations, partly avoiding the blockade of GABAergic transmission present with other caged compounds. Two-photon uncaging of RuBi-Glutamate has a high spatial resolution and generates excitatory responses in individual dendritic spines with physiological kinetics. With laser beam multiplexing, two-photon RuBi-Glutamate uncaging can also be used to depolarize and fi re pyramidal neurons with single-cell resolution. RuBi-Glutamate therefore enables the photoactivation of neuronal dendrites and circuits with visible or two-photon light sources, achieving single cell, or even single spine, precision.

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.DOI: http://dx.doi.org/10.7554/eLife.14371.001

In mammalian cortex, most excitatory inputs occur on dendritic spines, avoiding dendritic shafts. Although spines biochemically isolate inputs, nonspiny neurons can also implement biochemical compartmentalization; so, it is possible that spines have an additional function. We have recently shown that the spine neck can filter membrane potentials going into and out of the spine. To investigate the potential function of this electrical filtering, we used two-photon uncaging of glutamate and compared the integration of electrical signals in spines vs. dendritic shafts from basal dendrites of mouse layer 5 pyramidal neurons. Uncaging potentials onto spines summed linearly, whereas potentials on dendritic shafts reduced each other's effect. Linear integration of spines was maintained regardless of the amplitude of the response, distance between spines (as close as <2 m), distance of the spines to the soma, dendritic diameter, or spine neck length. Our findings indicate that spines serve as electrical isolators to prevent input interaction, and thus generate a linear arithmetic of excitatory inputs. Linear integration could be an essential feature of cortical and other spine-laden circuits.second harmonic ͉ pyramidal cell ͉ glutamate uncaging ͉ two-photon I n neocortex and many other brain areas, most excitatory inputs terminate on dendritic spines (1); so, spines must therefore likely be of major importance for the functioning of neural circuits (2). Spines can compartmentalize calcium (3), partly because their peculiar morphologies, with a small head separated from the dendrite by a slender neck, enable the biochemical isolation between inputs (4-6). This compartmentalization is thought to underlie input-specific forms of synaptic plasticity, such as long-term potentiation (7-9).Theoretical work spanning several decades has suggested that spines are ideally poised to play a major role in altering the electrical properties of synaptic inputs (2, 10 -14). Indeed, recent work has called into question the view that the sole function of spines is one of biochemical compartmentalization. First, nonspiny neurons can compartmentalize calcium with as good a degree of biochemical isolation between inputs as spiny cells (15,16). Also, by using glutamate uncaging and second harmonic measurements of membrane potential on spines of layer 5 pyramidal neurons, we have demonstrated that the spine neck filters membrane potentials (17). The filtering was bidirectional, i.e., both spine potentials transmitted to the dendrite and dendritic potentials transmitted to the spine were strongly attenuated. This implies that spines could isolate inputs electrically, an idea previously suggested based on theoretical calculations (11,12,18). More generally, passive cable models predict that inputs onto dendrites will shunt each other if they are close (19,20). Therefore, dendritic spines could provide an electrically isolated postsynaptic region to prevent interaction between different excitatory inputs, resulting in a linear integration (21).Co...

The structural organization of excitatory inputs supporting spike-timing-dependent plasticity (STDP) remains unknown. We performed a spine STDP protocol using two-photon (2P) glutamate uncaging (pre) paired with postsynaptic spikes (post) in layer 5 pyramidal neurons from juvenile mice. Here we report that pre-post pairings that trigger timing-dependent LTP (t-LTP) produce shrinkage of the activated spine neck and increase in synaptic strength; and post-pre pairings that trigger timing-dependent LTD (t-LTD) decrease synaptic strength without affecting spine shape. Furthermore, the induction of t-LTP with 2P glutamate uncaging in clustered spines (<5 μm apart) enhances LTP through a NMDA receptormediated spine calcium accumulation and actin polymerization-dependent neck shrinkage, whereas t-LTD was dependent on NMDA receptors and disrupted by the activation of clustered spines but recovered when separated by >40 μm. These results indicate that synaptic cooperativity disrupts t-LTD and extends the temporal window for the induction of t-LTP, leading to STDP only encompassing LTP.

We introduce a novel caged dopamine compound (RuBi-Dopa) based on ruthenium photochemistry. RuBi-Dopa has a high uncaging efficiency and can be released with visible (blue-green) and IR light in a two-photon regime. We combine two-photon photorelease of RuBi-Dopa with two-photon calcium imaging for an optical imaging and manipulation of dendritic spines in living brain slices, demonstrating that spines can express functional dopamine receptors. This novel compound allows mapping of functional dopamine receptors in living brain tissue with exquisite spatial resolution.