Functional and structural features of L2/3 pyramidal cells continuously covary with pial depth in mouse visual cortex.

Pyramidal cells of neocortical layer 2/3 (L2/3 PyrCs) integrate signals from numerous brain areas and project throughout the neocortex. These PyrCs show pial depth-dependent functional and structural specializations, indicating participation in different functional microcircuits. However, whether these depth-dependent differences result from separable PyrC subtypes or whether their features display a continuum correlated with pial depth is unknown. Here, we assessed the stimulus selectivity, electrophysiological properties, dendritic morphology, and excitatory and inhibitory connectivity across the depth of L2/3 in the binocular visual cortex of mice. We find that the apical, but not the basal dendritic tree structure, varies with pial depth, which is accompanied by variation in subthreshold electrophysiological properties. Lower L2/3 PyrCs receive increased input from L4, while upper L2/3 PyrCs receive a larger proportion of intralaminar input. In vivo calcium imaging revealed a systematic change in visual responsiveness, with deeper PyrCs showing more robust responses than superficial PyrCs. Furthermore, deeper PyrCs are more driven by contralateral than ipsilateral eye stimulation. Importantly, the property value transitions are gradual, and L2/3 PyrCs do not display discrete subtypes based on these parameters. Therefore, L2/3 PyrCs' multiple functional and structural properties systematically correlate with their depth, forming a continuum rather than discrete subtypes.

Function of dendritic spines on hippocampal inhibitory neurons.

The majority of γ-aminobutyric acid (GABA)ergic interneurons have smooth dendrites with no or only few dendritic spines, but certain types of spiny GABAergic interneurons do actually contain substantial numbers of spines. The explanation for such spines has so far been purely structural: They increase the dendritic surface area and thus provide the opportunity to accommodate larger numbers of synapses. We reasoned that there may be specific functional properties for these spines and therefore, undertook to characterize interneuron spines functionally. We find a remarkable similarity to pyramidal cell spines: They receive excitatory synapses with calcium impermeable α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors, compartmentalize biochemical signals, and display activity-dependent morphological plasticity. Nevertheless, notable differences in spine density, neck length, and spine-dendrite coupling exist. Thus, dendritic spines on inhibitory interneurons have a number of important functional properties that go substantially beyond simply expanding the dendritic surface area. It therefore seems likely that spiny and aspiny interneurons may have very different roles in neural circuit function and plasticity.

Polygenic risk for schizophrenia converges on alternative polyadenylation as molecular mechanism underlying synaptic impairment.

Schizophrenia (SCZ) is a genetically heterogenous psychiatric disorder of highly polygenic nature. Correlative evidence from genetic studies indicate that the aggregated effects of distinct genetic risk factor combinations found in each patient converge onto common molecular mechanisms. To prove this on a functional level, we employed a reductionistic cellular model system for polygenic risk by differentiating induced pluripotent stem cells (iPSCs) from 104 individuals with high polygenic risk load and controls into cortical glutamatergic neurons (iNs). Multi-omics profiling identified widespread differences in alternative polyadenylation (APA) in the 3' untranslated region of many synaptic transcripts between iNs from SCZ patients and healthy donors. On the cellular level, 3'APA was associated with a reduction in synaptic density of iNs. Importantly, differential APA was largely conserved between postmortem human prefrontal cortex from SCZ patients and healthy donors, and strongly enriched for transcripts related to synapse biology. 3'APA was highly correlated with SCZ polygenic risk and affected genes were significantly enriched for SCZ associated common genetic variation. Integrative functional genomic analysis identified the RNA binding protein and SCZ GWAS risk gene PTBP2 as a critical trans-acting factor mediating 3'APA of synaptic genes in SCZ subjects. Functional characterization of PTBP2 in iNs confirmed its key role in 3'APA of synaptic transcripts and regulation of synapse density. Jointly, our findings show that the aggregated effects of polygenic risk converge on 3'APA as one common molecular mechanism that underlies synaptic impairments in SCZ.

Orientation and direction tuning align with dendritic morphology and spatial connectivity in mouse visual cortex.

The functional properties of neocortical pyramidal cells (PCs), such as direction and orientation selectivity in visual cortex, predominantly derive from their excitatory and inhibitory inputs. For layer 2/3 (L2/3) PCs, the detailed relationship between their functional properties and how they sample and integrate information across cortical space is not fully understood. Here, we study this relationship by combining functional in vivo two-photon calcium imaging, in vitro functional circuit mapping, and dendritic reconstruction of the same L2/3 PCs in mouse visual cortex. Our work reveals direct correlations between dendritic morphology and functional input connectivity and the orientation as well as direction tuning of L2/3 PCs. First, the apical dendritic tree is elongated along the postsynaptic preferred orientation, considering the representation of visual space in the cortex as determined by its retinotopic organization. Additionally, sharply orientation-tuned cells show a less complex apical tree compared with broadly tuned cells. Second, in direction-selective L2/3 PCs, the spatial distribution of presynaptic partners is offset from the soma opposite to the preferred direction. Importantly, although the presynaptic excitatory and inhibitory input distributions spatially overlap on average, the excitatory input distribution is spatially skewed along the preferred direction, in contrast to the inhibitory distribution. Finally, the degree of asymmetry is positively correlated with the direction selectivity of the postsynaptic L2/3 PC. These results show that the dendritic architecture and the spatial arrangement of excitatory and inhibitory presynaptic cells of L2/3 PCs play important roles in shaping their orientation and direction tuning.

Allosteric modulation of the presynaptic Ca2+ sensor for vesicle fusion.

Neurotransmitter release is triggered by an increase in the cytosolic Ca2+ concentration ([Ca2+]i), but it is unknown whether the Ca2+-sensitivity of vesicle fusion is modulated during synaptic plasticity. We investigated whether the potentiation of neurotransmitter release by phorbol esters, which target presynaptic protein kinase C (PKC)/munc-13 signalling cascades, exerts a direct effect on the Ca2+-sensitivity of vesicle fusion. Using direct presynaptic Ca2+-manipulation and Ca2+ uncaging at a giant presynaptic terminal, the calyx of Held, we show that phorbol esters potentiate transmitter release by increasing the apparent Ca2+-sensitivity of vesicle fusion. Phorbol esters potentiate Ca2+-evoked release as well as the spontaneous release rate. We explain both effects by an increased fusion 'willingness' in a new allosteric model of Ca2+-activation of vesicle fusion. In agreement with an allosteric mechanism, we observe that the classically high Ca2+ cooperativity in triggering vesicle fusion (approximately 4) is gradually reduced below 3 microM [Ca2+]i, reaching a value of <1 at basal [Ca2+]i. Our data indicate that spontaneous transmitter release close to resting [Ca2+]i is a consequence of an intrinsic property of the molecular machinery that mediates synaptic vesicle fusion.

Limited functional convergence of eye-specific inputs in the retinogeniculate pathway of the mouse.

Segregation of retinal ganglion cell (RGC) axons by type and eye of origin is considered a hallmark of dorsal lateral geniculate nucleus (dLGN) structure. However, recent anatomical studies have shown that neurons in mouse dLGN receive input from multiple RGC types of both retinae. Whether convergent input leads to relevant functional interactions is unclear. We studied functional eye-specific retinogeniculate convergence using dual-color optogenetics in vitro. dLGN neurons were strongly dominated by input from one eye. Most neurons received detectable input from the non-dominant eye, but this input was weak, with a prominently reduced AMPAR:NMDAR ratio. Consistent with this, only a small fraction of thalamocortical neurons was binocular in vivo across visual stimuli and cortical projection layers. Anatomical overlap between RGC axons and dLGN neuron dendrites alone did not explain the strong bias toward monocularity. We conclude that functional eye-specific input selection and refinement limit convergent interactions in dLGN, favoring monocularity.

Inducing Different Neuronal Subtypes from Astrocytes in the Injured Mouse Cerebral Cortex.

Astrocytes are particularly promising candidates for reprogramming into neurons, as they maintain some of the original patterning information from their radial glial ancestors. However, to which extent the position of astrocytes influences the fate of reprogrammed neurons remains unknown. To elucidate this, we performed stab wound injury covering an entire neocortical column, including the gray matter (GM) and white matter (WM), and targeted local reactive astrocytes via injecting FLEx switch (Cre-On) adeno-associated viral (AAV) vectors into mGFAP-Cre mice. Single proneural factors were not sufficient for adequate reprogramming, although their combination with the nuclear receptor-related 1 protein (Nurr1) improved reprogramming efficiency. Nurr1 and Neurogenin 2 (Ngn2) resulted in high-efficiency reprogramming of targeted astrocytes into neurons that develop lamina-specific hallmarks, including the appropriate long-distance axonal projections. Surprisingly, in the WM, we did not observe any reprogrammed neurons, thereby unveiling a crucial role of region- and layer-specific differences in astrocyte reprogramming.

Live cell imaging reveals 3'-UTR dependent mRNA sorting to synapses.

mRNA transport restricts translation to specific subcellular locations, which is the basis for many cellular functions. However, the precise process of mRNA sorting to synapses in neurons remains elusive. Here we use Rgs4 mRNA to investigate 3'-UTR-dependent transport by MS2 live-cell imaging. The majority of observed RNA granules display 3'-UTR independent bidirectional transport in dendrites. Importantly, the Rgs4 3'-UTR causes an anterograde transport bias, which requires the Staufen2 protein. Moreover, the 3'-UTR mediates dynamic, sustained mRNA recruitment to synapses. Visualization at high temporal resolution enables us to show mRNA patrolling dendrites, allowing transient interaction with multiple synapses, in agreement with the sushi-belt model. Modulation of neuronal activity by either chemical silencing or local glutamate uncaging regulates both the 3'-UTR-dependent transport bias and synaptic recruitment. This dynamic and reversible mRNA recruitment to active synapses would allow translation and synaptic remodeling in a spatially and temporally adaptive manner.

Quantitative Analysis of the Spatial Organization of Synaptic Inputs on the Postsynaptic Dendrite.

The spatial organization of synaptic inputs on the dendritic tree of cortical neurons is considered to play an important role in the dendritic integration of synaptic activity. Active electrical properties of dendrites and mechanisms of dendritic integration have been studied for a long time. New technological developments are now enabling the characterization of the spatial organization of synaptic inputs on dendrites. However, quantitative methods for the analysis of such data are lacking. In order to place cluster parameters into the framework of dendritic integration and synaptic summation, these parameters need to be assessed rigorously in a quantitative manner. Here I present an approach for the analysis of synaptic input clusters on the dendritic tree that is based on combinatorial analysis of the likelihoods to observe specific input arrangements. This approach is superior to the commonly applied analysis of nearest neighbor distances between synaptic inputs comparing their distribution to simulations with random reshuffling or bootstrapping. First, the new approach yields exact likelihood values rather than approximate numbers obtained from simulations. Second and more importantly, the new approach identifies individual clusters and thereby allows to quantify and characterize individual cluster properties.

High-yield in vitro recordings from neurons functionally characterized in vivo.

In vivo two-photon calcium imaging provides detailed information about the activity and response properties of individual neurons. However, in vitro methods are often required to study the underlying neuronal connectivity and physiology at the cellular and synaptic levels at high resolution. This protocol provides a fast and reliable workflow for combining the two approaches by characterizing the response properties of individual neurons in mice in vivo using genetically encoded calcium indicators (GECIs), followed by retrieval of the same neurons in brain slices for further analysis in vitro (e.g., circuit mapping). In this approach, a reference frame is provided by fluorescent-bead tracks and sparsely transduced neurons expressing a structural marker in order to re-identify the same neurons. The use of GECIs provides a substantial advancement over previous approaches by allowing for repeated in vivo imaging. This opens the possibility of directly correlating experience-dependent changes in neuronal activity and feature selectivity with changes in neuronal connectivity and physiology. This protocol requires expertise both in in vivo two-photon calcium imaging and in vitro electrophysiology. It takes 3 weeks or more to complete, depending on the time allotted for repeated in vivo imaging of neuronal activity.

Nonlinear [Ca2+] signaling in dendrites and spines caused by activity-dependent depression of Ca2+ extrusion.

Spine Ca2+ triggers the induction of synaptic plasticity and other adaptive neuronal responses. The amplitude and time course of Ca2+ signals specify the activation of the signaling pathways that trigger different forms of plasticity such as long-term potentiation and depression. The shapes of Ca2+ signals are determined by the dynamics of Ca2+ sources, Ca2+ buffers, and Ca2+ extrusion mechanisms. Here we show in rat CA1 pyramidal neurons that plasma membrane Ca2+ pumps (PMCAs) and Na+/Ca2+ exchangers are the major Ca2+ extrusion pathways in spines and small dendrites. Surprisingly, we found that Ca2+ extrusion via PMCA and Na+/Ca2+ exchangers slows in an activity-dependent manner, mediated by intracellular Na+ and Ca2+ accumulations. This activity-dependent depression of Ca2+ extrusion is, in part, attributable to Ca2+-dependent inactivation of PMCAs. Ca2+ extrusion recovers from depression with a time constant of 0.5 s. Depression of Ca2+ extrusion provides a positive feedback loop, converting small differences in stimuli into large differences in Ca2+ concentration. Depression of Ca2+ extrusion produces Ca2+ concentration dynamics that depend on the history of neuronal activity and therefore likely modulates the induction of synaptic plasticity.

NMDA receptor subunit-dependent [Ca2+] signaling in individual hippocampal dendritic spines.

Ca2+ influx through synaptic NMDA receptors (NMDA-Rs) triggers a variety of adaptive cellular processes. To probe NMDA-R-mediated [Ca2+] signaling, we used two-photon glutamate uncaging to stimulate NMDA-Rs on individual dendritic spines of CA1 pyramidal neurons in rat brain slices. We measured NMDA-R currents at the soma and NMDA-R-mediated [Ca2+] transients in stimulated spines (Delta[Ca2+]). Uncaging-evoked NMDA-R current amplitudes were independent of the size of the stimulated spine, implying that smaller spines contain higher densities of functional NMDA-Rs. The ratio of Delta[Ca2+] over NMDA-R current was highly variable (factor of 10) across spines, especially for small spines. These differences were not explained by heterogeneity in spine sizes or diffusional coupling between spines and their parent dendrites. In addition, we find that small spines have NMDA-R currents that are sensitive to NMDA-R NR2B subunit-specific antagonists. With block of NR2B-containing receptors, the range of Delta[Ca2+]/NMDA-R current ratios and their average value were much reduced. Our data suggest that individual spines can regulate the subunit composition of their NMDA-Rs and the effective fractional Ca2+ current through these receptors.

Clusters of synaptic inputs on dendrites of layer 5 pyramidal cells in mouse visual cortex.

The spatial organization of synaptic inputs on the dendritic tree of cortical neurons plays a major role for dendritic integration and neural computations, yet, remarkably little is known about it. We mapped the spatial organization of glutamatergic synapses between layer 5 pyramidal cells by combining optogenetics and 2-photon calcium imaging in mouse neocortical slices. To mathematically characterize the organization of inputs we developed an approach based on combinatorial analysis of the likelihoods of specific synapse arrangements. We found that the synapses of intralaminar inputs form clusters on the basal dendrites of layer 5 pyramidal cells. These clusters contain 4 to 14 synapses within ≤30 µm of dendrite. According to the spatiotemporal characteristics of synaptic summation, these numbers suggest that there will be non-linear dendritic integration of synaptic inputs during synchronous activation.

Separation of presynaptic and postsynaptic contributions to depression by covariance analysis of successive EPSCs at the calyx of Held synapse.

Synaptic short-term plasticity is considered to result from multiple cellular mechanisms, which may include presynaptic and postsynaptic contributions. We have recently developed a nonstationary EPSC fluctuation analysis (Scheuss and Neher, 2001) to estimate synaptic parameters and their transient changes during short-term synaptic plasticity. Extending the classical variance-mean approach, a short train of stimuli is applied repetitively, and the resulting EPSCs are analyzed for means, variances, and covariances. This provides estimates of the quantal size and quantal content for each EPSC in the train, and furthermore, an estimate of the number of release sites. The latter is less sensitive to heterogeneity in the release probability than that of the variance-mean approach. Here, we applied this analysis to the calyx of Held synapse in brainstem slices of young rats (postnatal day 8-10). We found significant negative covariance in the amplitude of successive EPSCs in a train. The analysis showed that the 10-fold depression in the EPSC amplitude during 100 Hz trains at elevated extracellular Ca(2+) concentration resulted from a 2.5-fold reduction in quantal size caused by postsynaptic AMPA receptor desensitization and saturation, and a fourfold reduction in quantal content, which was partially relieved by application of cyclothiazide. The number of release sites estimated by covariance analysis was approximately 2000 and significantly larger than estimates from variance-mean parabolas.

Imaging calcium concentration dynamics in small neuronal compartments.

Calcium and its regulation play central roles diverse physiologic processes. Quantification of calcium concentrations ([Ca2+]) in small neuronal compartments is crucial to understanding Ca2+-dependent signaling. Here, we describe techniques that are optimized for 2-photon imaging of [Ca2+] dynamics in small compartments such as dendrites and dendritic spines.

Balance and stability of synaptic structures during synaptic plasticity.

Subsynaptic structures such as bouton, active zone, postsynaptic density (PSD) and dendritic spine, are highly correlated in their dimensions and also correlate with synapse strength. Why this is so and how such correlations are maintained during synaptic plasticity remains poorly understood. We induced spine enlargement by two-photon glutamate uncaging and examined the relationship between spine, PSD, and bouton size by two-photon time-lapse imaging and electron microscopy. In enlarged spines the PSD-associated protein Homer1c increased rapidly, whereas the PSD protein PSD-95 increased with a delay and only in cases of persistent spine enlargement. In the case of nonpersistent spine enlargement, the PSD proteins remained unchanged or returned to their original level. The ultrastructure at persistently enlarged spines displayed matching dimensions of spine, PSD, and bouton, indicating their correlated enlargement. This supports a model in which balancing of synaptic structures is a hallmark for the stabilization of structural modifications during synaptic plasticity.

Loss of sensory input causes rapid structural changes of inhibitory neurons in adult mouse visual cortex.

A fundamental property of neuronal circuits is the ability to adapt to altered sensory inputs. It is well established that the functional synaptic changes underlying this adaptation are reflected by structural modifications in excitatory neurons. In contrast, the degree to which structural plasticity in inhibitory neurons accompanies functional changes is less clear. Here, we use two-photon imaging to monitor the fine structure of inhibitory neurons in mouse visual cortex after deprivation induced by retinal lesions. We find that a subset of inhibitory neurons carry dendritic spines, which form glutamatergic synapses. Removal of visual input correlates with a rapid and lasting reduction in the number of inhibitory cell spines. Similar to the effects seen for dendritic spines, the number of inhibitory neuron boutons dropped sharply after retinal lesions. Together, these data suggest that structural changes in inhibitory neurons may precede structural changes in excitatory circuitry, which ultimately result in functional adaptation following sensory deprivation.

Rapid functional maturation of nascent dendritic spines.

Spine growth and retraction with synapse formation and elimination plays an important role in shaping brain circuits during development and in the adult brain, yet the temporal relationship between spine morphogenesis and the formation of functional synapses remains poorly defined. We imaged hippocampal pyramidal neurons to identify spines of different ages. We then used two-photon glutamate uncaging, whole-cell recording, and Ca(2+) imaging to analyze the properties of nascent spines and their older neighbors. New spines expressed glutamate-sensitive currents that were indistinguishable from mature spines of comparable volumes. Some spines exhibited negligible AMPA receptor-mediated responses, but the occurrence of these "silent" spines was uncorrelated with spine age. In contrast, NMDA receptor-mediated Ca(2+) accumulations were significantly lower in new spines. New spines reconstructed using electron microscopy made synapses. Our data support a model in which outgrowth and enlargement of nascent spines is tightly coupled to formation and maturation of glutamatergic synapses.

Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators.

Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.

Release kinetics, quantal parameters and their modulation during short-term depression at a developing synapse in the rat CNS.

We have characterized developmental changes in the kinetics and quantal parameters of action potential (AP)-evoked neurotransmitter release during maturation of the calyx of Held synapse. Quantal size (q) and peak amplitudes of evoked EPSCs increased moderately, whereas the fraction of vesicles released by single APs decreased. During synaptic depression induced in postnatal day (P) 5-7 synapses by 10-100 Hz stimulation, q declined rapidly to 40-12% of its initial value. The decrease in q was generally smaller in more mature synapses (P12-14), but quite severe for frequencies > or = 300 Hz. The stronger decline of q in immature synapses resulted from a slower recovery from desensitization, presumably due to delayed glutamate clearance. Recovery from this desensitization followed an exponential time course with a time constant of approximately 480 ms in P5-7 synapses, and sped up > 20-fold during maturation. Deconvolution analysis of EPSCs revealed a significant acceleration of the release time course during development, which was accompanied by a 2-fold increase of the peak release rate. During long 100 Hz trains, more mature synapses were able to sustain average rates of 8-10 quanta s(-1) per active zone for phasic release. The rates of asynchronous vesicle release increased transiently > 35-fold immediately after such stimuli and decayed rapidly with an exponential time constant of approximately 50 ms to low resting levels of spontaneous release. However, even following extended periods of 100 Hz stimulation, the amount of asynchronous release was relatively minor with peak rates of less than 5% of the average rate of synchronous release measured at steady state during the tetani. Therefore, a multitude of mechanisms seems to converge on the generation of fast, temporally precise and reliable high-frequency transmission at the mature calyx of Held synapse.