Alanna J. Watt

Activity Coregulates Quantal AMPA and NMDA Currents at Neocortical Synapses

AMPA and NMDA receptors are coexpressed at many central synapses, but the factors that control the ratio of these two receptors are not well understood. We recorded mixed miniature or evoked synaptic currents arising from coactivation of AMPA and NMDA receptors and found that long-lasting changes in activity scaled both currents up and down proportionally through changes in the number of postsynaptic receptors. The ratio of NMDA to AMPA current was similar at different synapses onto the same neuron, and this relationship was preserved following activity-dependent synaptic scaling. These data show that AMPA and NMDA receptors are tightly coregulated by activity at synapses at which they are both expressed and suggest that a mechanism exists to actively maintain a constant receptor ratio across a neurons synapses.

A proportional but slower NMDA potentiation follows AMPA potentiation in LTP

Most excitatory glutamatergic synapses contain both AMPA and NMDA receptors, but whether these receptors are regulated together or independently during synaptic plasticity has been controversial. Although long-term potentiation (LTP) is thought to selectively enhance AMPA currents and alter the NMDA-to-AMPA ratio, this ratio is well conserved across synapses onto the same neuron. This suggests that the NMDA-to-AMPA ratio is only transiently perturbed by LTP. To test this, we induced LTP at rat neocortical synapses and recorded mixed AMPA-NMDA currents. We observed rapid LTP of AMPA currents, as well as delayed potentiation of NMDA currents that required previous AMPA potentiation. The delayed potentiation of NMDA currents restored the original NMDA-to-AMPA ratio within 2 h of LTP induction. These data suggest that recruitment of AMPA receptors to synapses eventually induces a proportional increase in NMDA current. This may ensure that LTP does not alter the relative contributions of these two receptors to synaptic transmission and information processing.

Neuronal morphometry directly from bitmap images.

To the Editor: Neuroscientists measure the tree-like structures of neurons in order to better understand how neural circuits are constructed and how neural information is processed. In 1953, Donald Sholl published his well-known technique for quantitative analysis of the complex arbors of dendrites and axons1, but conventional methods still require reconstruction of arbors via time-consuming manual or semi-automated tracing from microscopy images. To bypass this reconstruction step and perform the Sholl technique directly on images instead, we developed Sholl Analysis (http://fiji.sc/Sholl), an open-source program for ImageJ/Fiji2 (Supplementary Fig. 1). The plug-in employs an improved algorithm to retrieve data from twoor three-dimensional (2D or 3D) bitmap images in any format supported by the Bio-Formats library (Supplementary Methods). It pairs this data retrieval with curve-fitting, regression analysis and statistical inference so that users can automatically extract a collection of Sholl-based metrics of arborization1,3 (Supplementary Note). Using individual cortical pyramidal neurons in 3D images, we found Sholl Analysis to be accurate when benchmarked against corresponding manual reconstructions (Supplementary Fig. 2). The method was also resilient to image degradation by simulated shot noise (Supplementary Fig. 3 and Supplementary Software). To further assess accuracy, and to explore the utility of Sholl Analysis in tackling neurons that are particularly slow to reconstruct manually, we studied cerebellar Purkinje cells in mice, which have large and intricate dendritic arbors. From tiled 3D image stacks of cerebellum (Fig. 1a), we selected seven Brainbow2.1-expressing Purkinje neurons and isolated their morphologies (Fig. 1b and Supplementary Note). We then used the Sholl Analysis software to retrieve ten metrics and found they were indistinguishable from those retrieved from manual reconstructions of the same 7 cells (Fig. 1c,d and Supplementary Methods). To probe the sensitivity of the Sholl Analysis software, we asked whether its metrics could be used to distinguish closelyrelated neocortical interneuron subtypes. Parvalbumin-positive (PV) interneurons in layer 5 of visual cortex can be morphologically classified into two subtypes on the basis of their axonal morphology: type 1 PV cells have ascending axons arborizing in layer 2/3, whereas axons of type 2 cells remain in layer 5 (ref. 4). Because their dendritic arbors are indistinguishable4, these two cell types otherwise appear highly similar (Fig. 1e,f). Using the Sholl Analysis software, we retrieved 18 metrics directly from 3D image stacks of 12 PV interneurons. We then used Ward’s hierarchical clustering based on these metrics to independently classify these cells (Fig. 1g and Supplementary Fig. 4). The 12 cells segregated into two groups: one group of five neurons and another of seven. We found that all the neurons but two were correctly classified, with one cell assigned incorrectly to each class (Fig. 1g). Thus, our use of the Sholl Analysis software to quantify arborization directly from bitmap images correctly identified 80–86% of cells. In agreement, linear Sholl plots of type 1 cells indicated more branching than was found for type 2 cells at a distance of 225–300 μm from the soma (Fig. 1h), which corresponds to check and inviting routine use. Second, the software can generate a summary report of the current system performance or a full report containing all individual PSF measurements and associated fitting parameters. Third, a table with the extracted resolution, planarity and colocalization data can be exported. This can be used for subsequent analysis, such as in an image processing or restoration pipeline. In addition, an average PSF from a user-selectable region of interest can be exported, for example, for image deconvolution. We used PSFj to quantify the performance of various high– numerical aperture (NA) objectives and to track day-to-day and system-to-system variation. The results showed substantial performance differences and allowed us to identify strengths and weaknesses of individual objectives as well as general shortcomings (Supplementary Figs. 1 and 2). In particular, we found that whereas lateral resolution performance generally fell short (~20–30%), axial resolution often met or exceeded expectations from the scalar approximation of the PSF commonly used in textbooks2 (Supplementary Note). Planarity was usually well corrected with variations over the FOV below the axial resolution and allowed for the detection of tilted slides caused, for example, by dust particles or misaligned slide holders or stages. Axial chromatic shifts were usually small, with little variation across the FOV (Supplementary Table 1). In contrast, chromatic shifts often showed circular symmetry and increased toward the edge of the FOV, which is a sign of lateral chromatic aberrations. Day-to-day performance variation of most objectives was relatively small (~2–6%) and comparable to single-measurement FOV variations (Supplementary Table 2). Furthermore, testing a limited number of identical objectives identified objective-toobjective and microscope-to-microscope variations of about 10% (Supplementary Tables 3 and 4). The PSFj software is open source and based on libraries from various sources, including ImageJ3 and μManager4, and it runs as a stand-alone application on the three major operating systems (using Java).

Activity-dependent remodeling of presynaptic inputs by postsynaptic expression of activated CaMKII.

Competitive synaptic remodeling is an important feature of developmental plasticity, but the molecular mechanisms remain largely unknown. Calcium/calmodulin-dependent protein kinase II (CaMKII) can induce postsynaptic changes in synaptic strength. We show that postsynaptic CaMKII also generates structural synaptic rearrangements between cultured cortical neurons. Postsynaptic expression of activated CaMKII (T286D) increased the strength of transmission between pairs of pyramidal neuron by a factor of 4, through a modest increase in quantal amplitude and a larger increase in the number of synaptic contacts. Concurrently, T286D reduced overall excitatory synaptic density and increased the proportion of unconnected pairs. This suggests that connectivity from some synaptic partners was increased while other partners were eliminated. The enhancement of connectivity required activity and NMDA receptor activation, while the elimination did not. These data suggest that postsynaptic activation of CaMKII induces a structural remodeling of presynaptic inputs that favors the retention of active presynaptic partners.

Homeostatic plasticity and STDP: keeping a neuron's cool in a fluctuating world

Spike-timing-dependent plasticity (STDP) offers a powerful means of forming and modifying neural circuits. Experimental and theoretical studies have demonstrated its potential usefulness for functions as varied as cortical map development, sharpening of sensory receptive fields, working memory, and associative learning. Even so, it is unlikely that STDP works alone. Unless changes in synaptic strength are coordinated across multiple synapses and with other neuronal properties, it is difficult to maintain the stability and functionality of neural circuits. Moreover, there are certain features of early postnatal development (e.g., rapid changes in sensory input) that threaten neural circuit stability in ways that STDP may not be well placed to counter. These considerations have led researchers to investigate additional types of plasticity, complementary to STDP, that may serve to constrain synaptic weights and/or neuronal firing. These are collectively known as “homeostatic plasticity” and include schemes that control the total synaptic strength of a neuron, that modulate its intrinsic excitability as a function of average activity, or that make the ability of synapses to undergo Hebbian modification depend upon their history of use. In this article, we will review the experimental evidence for homeostatic forms of plasticity and consider how they might interact with STDP during development, and learning and memory.

The metamorphosis of the developing cerebellar microcircuit.

Research highlights ► The developing cerebellar circuit exhibits transient synaptic elements. ► Network activity and synaptic plasticity are shaped by such transient circuit elements. ► Transient circuit features may be pivotal in the development of cerebellar circuits. ► The cerebellum is a highly attractive model system for the study of circuit development.

4-aminopyridine reverses ataxia and cerebellar firing deficiency in a mouse model of spinocerebellar ataxia type 6

Spinocerebellar ataxia type 6 (SCA6) is a devastating midlife-onset autosomal dominant motor control disease with no known treatment. Using a hyper-expanded polyglutamine (84Q) knock-in mouse, we found that cerebellar Purkinje cell firing precision was degraded in heterozygous (SCA684Q/+) mice at 19 months when motor deficits are observed. Similar alterations in firing precision and motor control were observed at disease onset at 7 months in homozygous (SCA684Q/84Q) mice, as well as a reduction in firing rate. We further found that chronic administration of the FDA-approved drug 4-aminopyridine (4-AP), which targets potassium channels, alleviated motor coordination deficits and restored cerebellar Purkinje cell firing precision to wildtype (WT) levels in SCA684Q/84Q mice both in acute slices and in vivo. These results provide a novel therapeutic approach for treating ataxic symptoms associated with SCA6.

Rapid Onset of Motor Deficits in a Mouse Model of Spinocerebellar Ataxia Type 6 Precedes Late Cerebellar Degeneration

Abstract Spinocerebellar ataxia type 6 (SCA6) is an autosomal-dominant cerebellar ataxia that has been associated with loss of cerebellar Purkinje cells. Disease onset is typically at midlife, although it can vary widely from late teens to old age in SCA6 patients. Our study focused on an SCA6 knock-in mouse model with a hyper-expanded (84X) CAG repeat expansion that displays midlife-onset motor deficits at ∼7 months old, reminiscent of midlife-onset symptoms in SCA6 patients, although a detailed phenotypic analysis of these mice has not yet been reported. Here, we characterize the onset of motor deficits in SCA684Q mice using a battery of behavioral assays to test for impairments in motor coordination, balance, and gait. We found that these mice performed normally on these assays up to and including at 6 months, but motor impairment was detected at 7 months with all motor coordination assays used, suggesting that motor deficits emerge rapidly during a narrow age window in SCA684Q mice. In contrast to what is seen in SCA6 patients, the decrease in motor coordination was observed without alterations in gait. No loss of cerebellar Purkinje cells or striatal neurons were observed at 7 months, the age at which motor deficits were first detected, but significant Purkinje cell loss was observed in 2-year-old SCA684Q mice, arguing that Purkinje cell death does not significantly contribute to the early stages of SCA6.

Transient cerebellar alterations during development prior to obvious motor phenotype in a mouse model of spinocerebellar ataxia type 6

Spinocerebellar ataxia type 6 (SCA6) is a midlife‐onset neurodegenerative disease caused by a CACNA1A mutation; CACNA1A is also implicated in cerebellar development. We have previously shown that when disease symptoms are present in midlife in SCA684Q/84Q mice, cerebellar Purkinje cells spike with reduced rate and precision. In contrast, we find that during postnatal development (P10–13), SCA684Q/84Q Purkinje cells spike with elevated rate and precision. Although surplus climbing fibres are linked to ataxia in other mouse models, we found surplus climbing fibre inputs on developing (P10–13) SCA684Q/84Q Purkinje cells when motor deficits were not detected. Developmental alterations were transient and were no longer observed in weanling (P21–24) SCA684Q/84Q Purkinje cells. Our results suggest that changes in the developing cerebellar circuit can occur without detectable motor abnormalities, and that changes in cerebellar development may not necessarily persist into adulthood.

In Vitro Investigation of Synaptic Plasticity

A classical in vitro model for investigation of information storage in the brain is based on the acute hippocampal slice. Here, repeated high-frequency stimulation of excitatory Schaeffer collaterals making synapses onto pyramidal cells in the hippocampal CA1 region leads to strengthening of evoked field-recording responses-long-term potentiation (LTP)-in keeping with Hebbs postulate. This model remains tremendously influential for its reliability, specificity, and relative ease of use. More recent plasticity studies have explored various other brain regions including the neocortex, which often requires more laborious whole-cell recordings of synaptically connected pairs of neurons, to ensure that the identities of recorded cells are known. In addition, with this experimental approach, the spiking activity can be controlled with millisecond precision, which is necessary for the study of spike-timing-dependent plasticity (STDP). Here, we provide protocols for in vitro study of hippocampal CA1 LTP using field recordings, and of STDP in synaptically connected pairs of layer-5 pyramidal cells in acute slices of rodent neocortex.