Cocaine Selectively Reorganizes Excitatory Inputs to Substantia Nigra Pars Compacta Dopamine Neurons.

Substantia nigra pars compacta (SNc) dopamine neurons and their targets are involved in addiction and cue-induced relapse. However, afferents onto SNc dopamine neurons themselves appear insensitive to drugs of abuse, such as cocaine, when afferents are collectively stimulated electrically. This contrasts with ventral tegmental area (VTA) dopamine neurons, whose glutamate afferents react robustly to cocaine. We used an optogenetic strategy to isolate identified SNc inputs and determine whether cocaine sensitivity in the mouse SNc circuit is conferred at the level of three glutamate afferents: dorsal raphé nucleus (DR), pedunculopontine nucleus (PPN), and subthalamic nucleus (STN). We found that excitatory afferents to SNc dopamine neurons are sensitive to cocaine in an afferent-specific manner. A single exposure to cocaine in vivo led to PPN-innervated synapses reducing the AMPA-to-NMDA receptor-mediated current ratio. In contrast to work in the VTA, this was due to increased NMDA receptor function with no change in AMPA receptor function. STN synapses showed a decrease in calcium-permeable AMPA receptors after cocaine, but no change in the AMPA-to-NMDA ratio. Cocaine also increased the release probability at DR-innervated and STN-innervated synapses, quantified by decreases in paired-pulse ratios. However, release probability at PPN-innervated synapses remained unaffected. By examining identified inputs, our results demonstrate a functional distribution among excitatory SNc afferent nuclei in response to cocaine, and suggest a compelling architecture for differentiation and separate parsing of inputs within the nigrostriatal system.SIGNIFICANCE STATEMENT Prior studies have established that substantia nigra pars compacta (SNc) dopamine neurons are a key node in the circuitry that drives addiction and relapse, yet cocaine apparently has no effect on electrically stimulated excitatory inputs. Our study is the first to demonstrate the functional impact of a drug of abuse on synaptic mechanisms of identified afferents to the SNc. Optogenetic dissection of inputs originating from dorsal raphé, pedunculopontine, and subthalamic nuclei were tested for synaptic modifications following in vivo cocaine exposure. Our results demonstrate that cocaine differentially induces modifications to SNc synapses depending on input origin. This presents implications for understanding dopamine processing of motivated behavior; most critically, it indicates that dopamine neurons selectively modulate signal reception processed by afferent nuclei.

Afadin, a Ras/Rap effector that controls cadherin function, promotes spine and excitatory synapse density in the hippocampus.

Many molecules regulate synaptogenesis, but intracellular signaling pathways required for their functions are poorly understood. Afadin is a Rap-regulated, actin-binding protein that promotes cadherin complex assembly as well as binding many other cell adhesion molecules and receptors. To examine its role in mediating synaptogenesis, we deleted afadin (mllt1), using a conditional allele, in postmitotic hippocampal neurons. Consistent with its role in promoting cadherin recruitment, afadin deletion resulted in 70% fewer and less intense N-cadherin puncta with similar reductions of ß-catenin and αN-catenin puncta densities and 35% reduction in EphB2 puncta density. Its absence also resulted in 40% decreases in spine and excitatory synapse densities in the stratum radiatum of CA1, as determined by morphology, apposition of presynaptic and postsynaptic markers, and synaptic transmission. The remaining synapses appeared to function normally. Thus, afadin is a key intracellular signaling molecule for cadherin recruitment and is necessary for spine and synapse formation in vivo.

Con-nectin axons and dendrites.

Unlike adherens junctions, synapses are asymmetric connections, usually between axons and dendrites, that rely on various cell adhesion molecules for structural stability and function. Two cell types of adhesion molecules found at adherens junctions, cadherins and nectins, are thought to mediate homophilic interaction between neighboring cells. In this issue, Togashi et al. (see p. 141) demonstrate that the differential localization of two heterophilic interacting nectins mediates the selective attraction of axons and dendrites in cooperation with cadherins.

SynapseJ: An Automated, Synapse Identification Macro for ImageJ.

While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a pre-synaptic marker, bassoon, with two different post-synaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.

Dorsal Raphe Dual Serotonin-Glutamate Neurons Drive Reward by Establishing Excitatory Synapses on VTA Mesoaccumbens Dopamine Neurons.

Dorsal raphe (DR) serotonin neurons provide a major input to the ventral tegmental area (VTA). Here, we show that DR serotonin transporter (SERT) neurons establish both asymmetric and symmetric synapses on VTA dopamine neurons, but most of these synapses are asymmetric. Moreover, the DR-SERT terminals making asymmetric synapses on VTA dopamine neurons coexpress vesicular glutamate transporter 3 (VGluT3; transporter for accumulation of glutamate for its synaptic release), suggesting the excitatory nature of these synapses. VTA photoactivation of DR-SERT fibers promotes conditioned place preference, elicits excitatory currents on mesoaccumbens dopamine neurons, increases their firing, and evokes dopamine release in nucleus accumbens. These effects are blocked by VTA inactivation of glutamate and serotonin receptors, supporting the idea of glutamate release in VTA from dual DR SERT-VGluT3 inputs. Our findings suggest a path-specific input from DR serotonergic neurons to VTA that promotes reward by the release of glutamate and activation of mesoaccumbens dopamine neurons.

Mosaic cellular patterning in the nose: Adhesion molecules give their two scents.

The sense of smell is mediated by the olfactory epithelium, which is composed of a mosaic pattern of olfactory sensory cells surrounded by supporting cells. In this issue, Katsunuma et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509020) show that the differential expression of nectins and cadherins establishes this pattern.

Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex.

The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine-coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3-4 h and neurons can be maintained in culture for up to 4 weeks.

Hairless and Wnt signaling: allies in epithelial stem cell differentiation.

Nuclear receptors and Wnt signaling are both important regulators of developmental and physiological processes. Recent work linking these pathways in epithelial stem cell differentiation has come from studies analyzing the in vivo function of the nuclear receptor corepressor, Hairless (HR). The HR protein has long been suspected to regulate a stem cell-mediated process, hair cycling, as mutations in the Hr gene cause hair loss in both mice and men. The discovery that the HR protein is a nuclear receptor corepressor indicated that HR function in hair cycling is by regulating gene expression. A recent study revealed that HR represses expression of Wise, an inhibitor of Wnt signaling, leading to a model in which HR controls the timing of Wnt signaling required for hair cycling. Here we review these data, and provide new data showing that HR corepressor activity is essential for its in vivo function, and identify an additional putative Wnt inhibitor regulated by HR. This work complements previous studies demonstrating the role of Wnt signaling in epithelial stem cell differentiation.

Hairless triggers reactivation of hair growth by promoting Wnt signaling.

The mammalian hair cycle involves periodic regeneration of a tiny organ, the hair follicle, through a stem-cell-mediated process. The Hairless (Hr) gene encodes a nuclear receptor corepressor (HR) that is essential for hair follicle regeneration, but its role in this process is unknown. Here, we demonstrate that transgenic expression of HR in progenitor keratinocytes rescues follicle regeneration in Hr(-/-) mice. We show that expression of Wise, a modulator of Wnt signaling, is repressed by HR in these cells, coincident with the timing of follicle regeneration. This work links HR and Wnt function, providing a model in which HR regulates the precise timing of Wnt signaling required for hair follicle regeneration.

The co-repressor hairless has a role in epithelial cell differentiation in the skin.

Although mutations in the mammalian hairless (Hr) gene result in congenital hair loss disorders in both mice and humans, the precise role of Hr in skin biology remains unknown. We have shown that the protein encoded by Hr (HR) functions as a nuclear receptor co-repressor. To address the role of HR in vivo, we generated a loss-of-function (Hr-/-) mouse model. The Hr-/- phenotype includes both hair loss and severe wrinkling of the skin. Wrinkling is correlated with increased cell proliferation in the epidermis and the presence of dermal cysts. In addition, a normally undifferentiated region, the infundibulum, is transformed into a morphologically distinct structure (utricle) that maintains epidermal function. Analysis of gene expression revealed upregulation of keratinocyte terminal differentiation markers and a novel caspase in Hr-/- skin, substantiating HR action as a co-repressor in vivo. Differences in gene expression occur prior to morphological changes in vivo, as well as in cultured keratinocytes, indicating that aberrant transcriptional regulation contributes to the Hr-/- phenotype. The properties of the cell types present in Hr-/- skin suggest that the normal balance of cell proliferation and differentiation is disrupted, supporting a model in which HR regulates the timing of epithelial cell differentiation in both the epidermis and hair follicle.