Stephanie Rudolph

Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca2+-permeable TRPC6 channels

The standardized extract of the St. Johns wort plant (Hypericum perforatum) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na+ concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca2+‐permeable, resulting in intracellular Ca2+ elevations. Indeed, hyperforin activates TRPC6‐mediated currents and Ca2+ transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca2+ transients and depolarizing inward currents sensitive to the TRPC channel blocker La3+, thus resembling the actions of the neurotrophin brain‐derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. Johns wort are mediated by a mechanism similar to that engaged by BDNF.

Spillover-Mediated Feedforward Inhibition Functionally Segregates Interneuron Activity

Neurotransmitter spillover represents a form of neural transmission not restricted to morphologically defined synaptic connections. Communication between climbing fibers (CFs) and molecular layer interneurons (MLIs) in the cerebellum is mediated exclusively by glutamate spillover. Here, we show how CF stimulation functionally segregates MLIs based on their location relative to glutamate release. Excitation of MLIs that reside within the domain of spillover diffusion coordinates inhibition of MLIs outside the diffusion limit. CF excitation of MLIs is dependent on extrasynaptic NMDA receptors that enhance the spatial and temporal spread of CF signaling. Activity mediated by functionally segregated MLIs converges onto neighboring Purkinje cells (PCs) to generate a long-lasting biphasic change in inhibition. These data demonstrate how glutamate release from single CFs modulates excitability of neighboring PCs, thus expanding the influence of CFs on cerebellar cortical activity in a manner not predicted by anatomical connectivity.

Cell type-specific manipulation with GFP-dependent Cre recombinase

There are many transgenic GFP reporter lines that allow the visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. We developed a method that exploits GFP for gene manipulation, Cre recombinase dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, which led to effective recombination selectively in GFP-labeled cells. Furthermore, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we found that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

Purkinje Cell Collaterals Enable Output Signals from the Cerebellar Cortex to Feed Back to Purkinje Cells and Interneurons

Purkinje cells (PCs) provide the sole output from the cerebellar cortex. Although PCs are well characterized on many levels, surprisingly little is known about their axon collaterals and their target neurons within the cerebellar cortex. It has been proposed that PC collaterals transiently control circuit assembly in early development, but it is thought that PC-to-PC connections are subsequently pruned. Here, we find that all PCs have collaterals in young, juvenile, and adult mice. Collaterals are restricted to the parasagittal plane, and most synapses are located in close proximity to PCs. Using optogenetics and electrophysiology, we find that in juveniles and adults, PCs make synapses onto other PCs, molecular layer interneurons, and Lugaro cells, but not onto Golgi cells. These findings establish that PC output can feed back and regulate numerous circuit elements within the cerebellar cortex and is well suited to contribute to processing in parasagittal zones.

Purkinje Cells Directly Inhibit Granule Cells in Specialized Regions of the Cerebellar Cortex

Inhibition of granule cells plays a key role in gating the flow of signals into the cerebellum, and it is thought that Golgi cells are the only interneurons that inhibit granule cells. Here we show that Purkinje cells, the sole output neurons of the cerebellar cortex, also directly inhibit granule cells via their axon collaterals. Anatomical and optogenetic studies indicate that this non-canonical feedback is region specific: it is most prominent in lobules that regulate eye movement and process vestibular information. Collaterals provide fast, slow, and tonic inhibition to granule cells, and thus allow Purkinje cells to regulate granule cell excitability on multiple timescales. We propose that this feedback mechanism could regulate excitability of the input layer, contribute to sparse coding, and mediate temporal integration.

Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies

The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. DOI: http://dx.doi.org/10.7554/eLife.15312.001

Active Dendrites and Differential Distribution of Calcium Channels Enable Functional Compartmentalization of Golgi Cells

Interneurons are essential to controlling excitability, timing, and synaptic integration in neuronal networks. Golgi cells (GoCs) serve these roles at the input layer of the cerebellar cortex by releasing GABA to inhibit granule cells (grcs). GoCs are excited by mossy fibers (MFs) and grcs and provide feedforward and feedback inhibition to grcs. Here we investigate two important aspects of GoC physiology: the properties of GoC dendrites and the role of calcium signaling in regulating GoC spontaneous activity. Although GoC dendrites are extensive, previous studies concluded they are devoid of voltage-gated ion channels. Hence, the current view holds that somatic voltage signals decay passively within GoC dendrites, and grc synapses onto distal dendrites are not amplified and are therefore ineffective at firing GoCs because of strong passive attenuation. Using whole-cell recording and calcium imaging in rat slices, we find that dendritic voltage-gated sodium channels allow somatic action potentials to activate voltage-gated calcium channels (VGCCs) along the entire dendritic length, with R-type and T-type VGCCs preferentially located distally. We show that R- and T-type VGCCs located in the dendrites can boost distal synaptic inputs and promote burst firing. Active dendrites are thus critical to the regulation of GoC activity, and consequently, to the processing of input to the cerebellar cortex. In contrast, we find that N-type channels are preferentially located near the soma, and control the frequency and pattern of spontaneous firing through their close association with calcium-activated potassium (KCa) channels. Thus, VGCC types are differentially distributed and serve specialized functions within GoCs. SIGNIFICANCE STATEMENT Interneurons are essential to neural processing because they modulate excitability, timing, and synaptic integration within circuits. At the input layer of the cerebellar cortex, a single type of interneuron, the Golgi cell (GoC), carries these functions. The extent of inhibition depends on both spontaneous activity of GoCs and the excitatory synaptic input they receive. In this study, we find that different types of calcium channels are differentially distributed, with dendritic calcium channels being activated by somatic activity, boosting synaptic inputs and enabling bursting, and somatic calcium cannels promoting regular firing. We therefore challenge the current view that GoC dendrites are passive and identify the mechanisms that contribute to GoCs regulating the flow of sensory information in the cerebellar cortex.

Location matters: somatic and dendritic SK channels answer to distinct calcium signals

Voltage-dependent calcium channels (VDCCs) couple neuronal activity to diverse intracellular signals with exquisite spatiotemporal specificity. Using calcium imaging and electrophysiology, Jones and Stuart (J Neurosci 33: 19396-19405, 2013) examined the intimate relationship between distinct types of VDCCs and small-conductance calcium-activated potassium (SK) channels that contribute to the compartmentalized control of excitability in the soma and dendrites of cortical pyramidal neurons. Here we discuss the importance of calcium domains for signal specificity, explore the possible functions and mechanisms for local control of SK channels, and highlight technical considerations for the optical detection of calcium signals.

Viral Delivery of GFP-Dependent Recombinases to the Mouse Brain

Many genetic tools have been developed that use green fluorescent protein (GFP) and its derivatives for labeling specific cell populations in organisms and in cell culture. To extend the use of GFP beyond labeling purposes, we developed methods and reagents that use GFP as a driver of biological activities. We used nanobodies that bind GFP to engineer CRE-DOG and Flp-DOG, recombinases that can induce Cre/lox and Flp/FRT recombination in a GFP-dependent manner, respectively. Here, we present a protocol to deliver CRE-DOG and Flp-DOG into the mouse brain by recombinant AAV infection. This protocol enables one to manipulate gene expression specifically in GFP-expressing cells, found either in transgenic GFP reporter lines or in cells made to express GFP by other transduction methods.

Sensitive Periods for Cerebellar-Mediated Autistic-like Behaviors

SUMMARY Despite a prevalence exceeding 1%, mechanisms underlying autism spectrum disorders (ASDs) are poorly understood, and targeted therapies and guiding parameters are urgently needed. We recently demonstrated that cerebellar dysfunction is sufficient to generate autistic-like behaviors in a mouse model of tuberous sclerosis complex (TSC). Here, using the mechanistic target of rapamycin (mTOR)-specific inhibitor rapamycin, we define distinct sensitive periods for treatment of autistic-like behaviors with sensitive periods extending into adulthood for social behaviors. We identify cellular and electro-physiological parameters that may contribute to behavioral rescue, with rescue of Purkinje cell survival and excitability corresponding to social behavioral rescue. In addition, using anatomic and diffusion-based MRI, we identify structural changes in cerebellar domains implicated in ASD that correlate with sensitive periods of specific autism-like behaviors. These findings thus not only define treatment parameters into adulthood, but also support a mechanistic basis for the targeted rescue of autism-related behaviors.