John Meitzen

Steroid Hormones Act Transsynaptically within the Forebrain to Regulate Neuronal Phenotype and Song Stereotypy

Steroid sex hormones induce dramatic seasonal changes in reproductive related behaviors and their underlying neural substrates in seasonally breeding vertebrates. For example, in adult white-crowned sparrows, increased Spring photoperiod raises circulating testosterone, causing morphological and electrophysiological changes in song-control nuclei, which modify song behavior for the breeding season. We investigated how photoperiod and steroid hormones induce these changes in morphology, electrophysiology, and behavior. Neurons in a song premotor nucleus, the robust nucleus of the arcopallium (RA), show increased intrinsic spontaneous firing rate and soma size when birds are in breeding condition. Using combinations of systemic and unilateral local intracerebral hormonal manipulations, we show that long-day photoperiod accelerates the effects of systemic testosterone on RA neurons via the estradiol-synthesizing enzyme aromatase (CYP19A1); these changes require inputs from the afferent song control nucleus HVC (used as a proper name) and steroid receptor activation within HVC; local coactivation of androgen and estrogen receptors (ARs and ERs, respectively) within HVC, but not RA, is sufficient to cause neuronal changes in RA; activation of ARs in RA is also permissive. Using bilateral local intracerebral hormone-receptor blockade, we found that ARs and ERs in the song-control nucleus HVC mediate systemic testosterone-induced changes in song stereotypy but not rate. This novel transsynaptic effect of gonadal steroids on activity and morphology of RA neurons is part of a concerted change in key premotor nuclei, enabling stereotyped song.

Estrogen receptors stimulate brain region specific metabotropic glutamate receptors to rapidly initiate signal transduction pathways

Estradiol and other steroid hormones modulate the nervous system and behavior on both acute and long-term time scales. Though estradiol was originally characterized as a regulator of gene expression through the action of nuclear estrogen receptors (ERs) that directly bind DNA, research over the past thirty years has firmly established that estradiol can bind to extra-nuclear ERs associated with the cellular membrane, producing changes in neurons through stimulation of various intracellular signaling pathways. Several studies have determined that the classical ERs, ERα and ERβ, mediate some of these fast-acting signaling pathways through activation of G proteins. Since ERα and ERβ are not G protein-coupled receptors, the mechanisms by which ERs can stimulate signal transduction pathways are a focus of recent research. Here we discuss recent studies illustrating one mechanism by which ERα and ERβ initiate these pathways: through direct association with metabotropic glutamate receptors (mGluRs). Estradiol binding to these membrane-localized estrogen receptors results in mGluR signaling independent of glutamate. ERs are organized with mGluRs into functional signaling microdomains via caveolin proteins. The pairing of ERs to specific mGluRs via caveolins is region specific, with ERs being linked to different mGluRs in hippocampal, striatal, and other neurons. It is becoming clear that ER signaling through mGluRs is one important mechanism by which estrogens can modulate neuron and glial physiology, ultimately impacting various aspects of nervous system function.

Palmitoylation of Estrogen Receptors Is Essential for Neuronal Membrane Signaling

In addition to activating nuclear estrogen receptor signaling, 17β-estradiol can also regulate neuronal function via surface membrane receptors. In various brain regions, these actions are mediated by the direct association of estrogen receptors (ERs) activating metabotropic glutamate receptors (mGluRs). These ER/mGluR signaling partners are organized into discrete functional microdomains via caveolin proteins. A central question that remains concerns the underlying mechanism by which these subpopulations of ERs are targeted to the surface membrane. One candidate mechanism is S-palmitoylation, a posttranscriptional modification that affects the subcellular distribution and function of the modified protein, including promoting localization to membranes. Here we test for the role of palmitoylation and the necessity of specific palmitoylacyltransferase proteins in neuronal membrane ER action. In hippocampal neurons, pharmacological inhibition of palmitoylation eliminated 17β-estradiol-mediated phosphorylation of cAMP response element-binding protein, a process dependent on surface membrane ERs. In addition, mutation of the palmitoylation site on estrogen receptor (ER) α blocks ERα-mediated cAMP response element-binding protein phosphorylation. Similar results were obtained after mutation of the palmitoylation site on ERβ. Importantly, mutation of either ERα or ERβ did not affect the ability of the reciprocal ER to signal at the membrane. In contrast, membrane ERα and ERβ signaling were both dependent on the expression of the palmitoylacyltransferase proteins DHHC-7 and DHHC-21. Neither mGluR activity nor caveolin or ER expression was affected by knockdown of DHHC-7 and DHHC-21. These data collectively suggest discrete mechanisms that regulate specific isoform or global membrane ER signaling in neurons separate from mGluR activity or nuclear ER function.

Plastic and Stable Electrophysiological Properties of Adult Avian Forebrain Song-Control Neurons across Changing Breeding Conditions

Steroid sex hormones drive changes in the nervous system and behavior in many animal taxa, but integrating the former with the latter remains challenging. One useful model system for meeting this challenge is seasonally breeding songbirds. In these species, plasma testosterone levels rise and fall across the seasons, altering song behavior and causing dramatic growth and regression of the song-control system, a discrete set of nuclei that control song behavior. Whereas the cellular mechanisms underlying changes in nucleus volume have been studied as a model for neural growth and degeneration, it is unknown whether these changes in neural structure are accompanied by changes in electrophysiological properties other than spontaneous firing rate. Here we test the hypothesis that passive and active neuronal properties in the forebrain song-control nuclei HVC and RA change across breeding conditions. We exposed adult male Gambels white-crowned sparrows to either short-day photoperiod or long-day photoperiod and systemic testosterone to simulate nonbreeding and breeding conditions, respectively. We made whole-cell recordings from RA and HVC neurons in acute brain slices. We found that RA projection neuron membrane time constant, capacitance, and evoked and spontaneous firing rates were all increased in the breeding condition; the measured electrophysiological properties of HVC interneurons and projection neurons were stable across breeding conditions. This combination of plastic and stable intrinsic properties could directly impact the song-control systems motor control across seasons, underlying changes in song stereotypy. These results provide a valuable framework for integrating how steroid hormones modulate cellular physiology to change behavior.

Seasonal changes in intrinsic electrophysiological activity of song control neurons in wild song sparrows

Song behavior and its underlying neural substrate can change seasonally in adult songbirds. To test whether environmental cues induce seasonal changes in electrophysiological characteristics of song control neurons, we measured in vitro intrinsic neuronal activity in the song control nucleus RA of adult male song sparrows (Melospiza melodia) in both the fall non-breeding and spring breeding seasons. We found that RA neurons in spring-captured birds show a more than threefold increase in spontaneous firing rate compared to those from fall-captured birds. We conclude that environmental cues are sufficient to induce seasonal changes in electrophysiological properties of song control neurons, and that changes in these properties may underlie seasonal changes in song behavior.

β1-Adrenergic receptors activate two distinct signaling pathways in striatal neurons

J. Neurochem. (2011) 116, 984–995.

Seasonal changes in patterns of gene expression in avian song control brain regions

Photoperiod and hormonal cues drive dramatic seasonal changes in structure and function of the avian song control system. Little is known, however, about the patterns of gene expression associated with seasonal changes. Here we address this issue by altering the hormonal and photoperiodic conditions in seasonally-breeding Gambels white-crowned sparrows and extracting RNA from the telencephalic song control nuclei HVC and RA across multiple time points that capture different stages of growth and regression. We chose HVC and RA because while both nuclei change in volume across seasons, the cellular mechanisms underlying these changes differ. We thus hypothesized that different genes would be expressed between HVC and RA. We tested this by using the extracted RNA to perform a cDNA microarray hybridization developed by the SoNG initiative. We then validated these results using qRT-PCR. We found that 363 genes varied by more than 1.5 fold (>log2 0.585) in expression in HVC and/or RA. Supporting our hypothesis, only 59 of these 363 genes were found to vary in both nuclei, while 132 gene expression changes were HVC specific and 172 were RA specific. We then assigned many of these genes to functional categories relevant to the different mechanisms underlying seasonal change in HVC and RA, including neurogenesis, apoptosis, cell growth, dendrite arborization and axonal growth, angiogenesis, endocrinology, growth factors, and electrophysiology. This revealed categorical differences in the kinds of genes regulated in HVC and RA. These results show that different molecular programs underlie seasonal changes in HVC and RA, and that gene expression is time specific across different reproductive conditions. Our results provide insights into the complex molecular pathways that underlie adult neural plasticity.

Time course of changes in Gambel's white-crowned sparrow song behavior following transitions in breeding condition

Seasonal changes in behavior and in its underlying neural substrate are common across animal taxa. These changes are often triggered by steroid sex hormones. Song in seasonally breeding songbirds provides an excellent example of this phenomenon. In these species, dramatic seasonal changes mediated by testosterone and its metabolites occur in adult song behavior and in the neural circuitry controlling song. While song rate can quickly change in response to seasonal breeding cues, it is unknown how quickly other aspects of song change, particularly the stereotypy of song phonology and syntax. In this study we determined whether and how quickly song rate, phonology, and syntax change in response to breeding and non-breeding physiological cues. We asked these questions using Gambels white-crowned sparrows (Zonotrichia leucophrys gambelii), a closed-ended learner with well-characterized changes in the neural circuitry controlling song behavior. We exposed ten photosensitive sparrows to long-day photoperiod and implanted them with subcutaneous testosterone pellets (day 0) to simulate breeding conditions. We continuously recorded song and found that song rate increased quickly, reaching maximum around day 6. The stereotypy of song phonology changed more slowly, reaching maximum by day 10 or later. Song syntax changed minimally after day 6, the earliest time point examined. After 21 days, we transitioned five birds from breeding to non-breeding condition. Song rate declined precipitously. These results suggest that while song rate changes quickly, song phonology changes more slowly, generally following or in parallel with previously investigated changes in the neural substrate.

Corticotropin releasing factor-induced CREB activation in striatal neurons occurs via a novel Gβγ signaling pathway.

The peptide corticotropin-releasing factor (CRF) was initially identified as a critical component of the stress response. CRF exerts its cellular effects by binding to one of two cognate G-protein coupled receptors (GPCRs), CRF receptor 1 (CRFR1) or 2 (CRFR2). While these GPCRs were originally characterized as being coupled to Gαs, leading to downstream activation of adenylyl cyclase (AC) and subsequent increases in cAMP, it has since become clear that CRFRs couple to and activate numerous other downstream signaling cascades. In addition, CRF signaling influences the activity of many diverse brain regions, affecting a variety of behaviors. One of these regions is the striatum, including the nucleus accumbens (NAc). CRF exerts profound effects on striatal-dependent behaviors such as drug addiction, pair-bonding, and natural reward. Recent data indicate that at least some of these behaviors regulated by CRF are mediated through CRF activation of the transcription factor CREB. Thus, we aimed to elucidate the signaling pathway by which CRF activates CREB in striatal neurons. Here we describe a novel neuronal signaling pathway whereby CRF leads to a rapid Gβγ- and MEK-dependent increase in CREB phosphorylation. These data are the first descriptions of CRF leading to activation of a Gβγ-dependent signaling pathway in neurons, as well as the first description of Gβγ activation leading to downstream CREB phosphorylation in any cellular system. Additionally, these data provide additional insight into the mechanisms by which CRF can regulate neuronal function.

Measurements of neuron soma size and density in rat dorsal striatum, nucleus accumbens core and nucleus accumbens shell: differences between striatal region and brain hemisphere, but not sex.

Both hemispheric bias and sex differences exist in striatal-mediated behaviors and pathologies. The extent to which these dimorphisms can be attributed to an underlying neuroanatomical difference is unclear. We therefore quantified neuron soma size and density in the dorsal striatum (CPu) as well as the core (AcbC) and shell (AcbS) subregions of the nucleus accumbens to determine whether these anatomical measurements differ by region, hemisphere, or sex in adult Sprague-Dawley rats. Neuron soma size was larger in the CPu than the AcbC or AcbS. Neuron density was greatest in the AcbS, intermediate in the AcbC, and least dense in the CPu. CPu neuron density was greater in the left in comparison to the right hemisphere. No attribute was sexually dimorphic. These results provide the first evidence that hemispheric bias in the striatum and striatal-mediated behaviors can be attributed to a lateralization in neuronal density within the CPu. In contrast, sexual dimorphisms appear mediated by factors other than gross anatomical differences.