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...