Sex differences in susceptibility to substance use disorder: Role for X chromosome inactivation and escape?

There is a sex-based disparity associated with substance use disorders (SUDs) as demonstrated by clinical and preclinical studies. Females are known to escalate from initial drug use to compulsive drug-taking behavior (telescoping) more rapidly, and experience greater negative withdrawal effects than males. Although these biological differences have largely been attributed to sex hormones, there is evidence for non-hormonal factors, such as the influence of the sex chromosome, which underlie sex disparities in addiction behavior. However, genetic and epigenetic mechanisms underlying sex chromosome influences on substance abuse behavior are not completely understood. In this review, we discuss the role that escape from X-chromosome inactivation (XCI) in females plays in sex-associated differences in addiction behavior. Females have two X chromosomes (XX), and during XCI, one X chromosome is randomly chosen to be transcriptionally silenced. However, some X-linked genes escape XCI and display biallelic gene expression. We generated a mouse model using an X-linked gene specific bicistronic dual reporter mouse as a tool to visualize allelic usage and measure XCI escape in a cell specific manner. Our results revealed a previously undiscovered X-linked gene XCI escaper (CXCR3), which is variable and cell type dependent. This illustrates the highly complex and context dependent nature of XCI escape which is largely understudied in the context of SUD. Novel approaches such as single cell RNA sequencing will provide a global molecular landscape and impact of XCI escape in addiction and facilitate our understanding of the contribution of XCI escape to sex disparities in SUD.

Biased Opioid Antagonists as Modulators of Opioid Dependence: Opportunities to Improve Pain Therapy and Opioid Use Management.

Opioid analgesics are effective pain therapeutics but they cause various adverse effects and addiction. For safer pain therapy, biased opioid agonists selectively target distinct µ opioid receptor (MOR) conformations, while the potential of biased opioid antagonists has been neglected. Agonists convert a dormant receptor form (MOR-µ) to a ligand-free active form (MOR-µ*), which mediates MOR signaling. Moreover, MOR-µ converts spontaneously to MOR-µ* (basal signaling). Persistent upregulation of MOR-µ* has been invoked as a hallmark of opioid dependence. Contrasting interactions with both MOR-µ and MOR-µ* can account for distinct pharmacological characteristics of inverse agonists (naltrexone), neutral antagonists (6ß-naltrexol), and mixed opioid agonist-antagonists (buprenorphine). Upon binding to MOR-µ*, naltrexone but not 6ß-naltrexol suppresses MOR-µ*signaling. Naltrexone blocks opioid analgesia non-competitively at MOR-µ*with high potency, whereas 6ß-naltrexol must compete with agonists at MOR-µ, accounting for ~100-fold lower in vivo potency. Buprenorphine's bell-shaped dose-response curve may also result from opposing effects on MOR-µ and MOR-µ*. In contrast, we find that 6ß-naltrexol potently prevents dependence, below doses affecting analgesia or causing withdrawal, possibly binding to MOR conformations relevant to opioid dependence. We propose that 6ß-naltrexol is a biased opioid antagonist modulating opioid dependence at low doses, opening novel avenues for opioid pain therapy and use management.

Circadian expression and functional characterization of PEA-15 within the mouse suprachiasmatic nucleus.

The circadian timing system influences the functional properties of most, if not all, physiological processes. Central to the mammalian timing system is the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN functions as a 'master clock' that sets the phasing of ancillary circadian oscillator populations found throughout the body. Further, via an entraining input from the retina, the SCN ensures that the clock oscillators are synchronized to the daily light/dark cycle. A critical component of the SCN timing and entrainment systems is the p44/42 mitogen-activated protein kinase (ERK/MAPK) pathway. Here, we examined the expression and function of phosphoprotein-enriched in astrocytes (PEA-15), an ERK scaffold protein that serves as a key regulator of MAPK signaling. A combination of immunolabeling and Western blotting approaches revealed high levels of PEA-15 within the SCN. PEA-15 expression was enriched in distinct subpopulations of SCN neurons, including arginine vasopressin (AVP)-positive neurons of the SCN shell region. Further, expression profiling detected a significant circadian oscillation in PEA-15 expression within the SCN. Brief photic stimulation during the early subjective night led to a significant increase in PEA-15 phosphorylation, an event that can trigger ERK/PEA-15 dissociation. Consistent with this, co-immunoprecipitation assays revealed that PEA-15 is directly bound to ERK in the SCN and that photic stimulation leads to their dissociation. Finally, we show that PEA-15 regulates ERK/MAPK-dependent activation of the core clock gene period1. Together, these data raise the prospect that PEA-15 functions as a key regulator of the SCN timing system.

Preferential Delivery of an Opioid Antagonist to the Fetal Brain in Pregnant Mice.

Prolonged fetal exposure to opioids results in neonatal abstinence syndrome (NAS), a major medical problem requiring intensive care and increased hospitalization times for newborns with NAS. Multiple strategies are currently available to alleviate withdrawal in infants with NAS. To prevent NAS caused by opioid maintenance programs in pregnant women, blocking fetal dependence without compromising the mother's opiate therapy is desirable. Here we tested in pregnant mice whether a peripherally selective opioid antagonist can preferentially enter the fetal brain and, thereby, in principle, selectively protect the fetus. We show using mass spectrometry that 6ß-naltrexol, a neutral opioid antagonist with very limited ability to cross the blood-brain barrier (BBB), readily crosses the placental barrier and enters the fetal brain at high levels, although it is relatively excluded from the maternal brain. Furthermore, owing to the late development of the BBB in postnatal mice, we show that 6ß-naltrexol can readily enter the juvenile mouse brain until at least postnatal day 14. Taking advantage of this observation, we show that long-term exposure to morphine starting in the second postnatal week causes robust and quantifiable dependence behaviors that are suppressed by concomitant administration of 6ß-naltrexol with much greater potency (ID50 0.022-0.044 mg/kg, or 1/500 the applied dose of morphine) than previously demonstrated for either the suppression of central nervous system opioid effects or the induction of withdrawal in adults. These results indicate that peripherally selective opioid antagonists capable of penetrating the placenta may be beneficial for preventing or reducing neonatal dependence and NAS in a dose range that should not interfere with maternal opioid maintenance.

p75 regulates Purkinje cell firing by modulating SK channel activity through Rac1.

p75 is expressed among Purkinje cells in the adult cerebellum, but its function has remained obscure. Here we report that p75 is involved in maintaining the frequency and regularity of spontaneous firing of Purkinje cells. The overall spontaneous firing activity of Purkinje cells was increased in p75(-/-) mice during the phasic firing period due to a longer firing period and accompanying reduction in silence period than in the wild type. We attribute these effects to a reduction in small conductance Ca(2+)-activated potassium (SK) channel activity in Purkinje cells from p75(-/-) mice compared with the wild type littermates. The mechanism by which p75 regulates SK channel activity appears to involve its ability to activate Rac1. In organotypic cultures of cerebellar slices, brain-derived neurotrophic factor increased RacGTP levels by activating p75 but not TrkB. These results correlate with a reduction in RacGTP levels in synaptosome fractions from the p75(-/-) cerebellum, but not in that from the cortex of the same animals, compared with wild type littermates. More importantly, we demonstrate that Rac1 modulates SK channel activity and firing patterns of Purkinje cells. Along with the finding that spine density was reduced in p75(-/-) cerebellum, these data suggest that p75 plays a role in maintaining normalcy of Purkinje cell firing in the cerebellum in part by activating Rac1 in synaptic compartments and modulating SK channels.

Whole transcriptome RNA-Seq allelic expression in human brain.

BACKGROUND: Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals. RESULTS: We find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals. CONCLUSIONS: These results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.

Sex-dependent behavioral functions of the Purkinje cell-specific Gαi/o binding protein, Pcp2(L7).

We previously reported motor and non-motor enhancements in a mouse mutant with an inactivated Purkinje cell-specific gene, Pcp2(L7), that encodes a GoLoco domain-containing modulator of Gi/o protein-coupled receptors. Effects included elevated learning asymptote with repeated rotarod training, increased acquisition rate in tone-cued fear conditioning (FC), and subtle male-specific changes in both acoustic startle habituation and pre-pulse inhibition. We have further analyzed this mutant strain this time with a focus on male-female differences, and here we report a sex-dependent anxiety-like phenotype: male mutants are less anxious, and female mutants are more anxious, than same-sex wild types. Similarly, the fear responses measured during the tone in FC acquisition are decreased in male mutants and increased in female mutants relative to same-sex wild types. Overall, the dynamics of both acquisition and extinction of FC is affected in mutants but memory was not affected. In the social realm, compositional analysis of sociability and preference for social novelty data supports that both L7 genotype and sex contribute to these behaviors. These results provide direct evidence of emotional functions of the cerebellum due to the unambiguous cerebellar specificity of Pcp2(L7) expression and the lack of any confounding motor defects in the mutant. We attempt to synthesize these new data with what is previously known both about Pcp2(L7) and about the effects of sex and sex hormones on anxiety and fear behaviors: specifically, L7 is a bidirectional and sex-dependent damper that regulates the amplitude and/or rate of sensorimotor responses, potentially acting as a mood stabilizer.

Cerebellar zones: history, development, and function.

The longitudinal and transverse zonal arrangement of axonal projections to and from the cerebellum, even more than the well-known laminar cytoarchitecture, is the hallmark of cerebellar anatomy. No model of cerebellar function, whether in motor control, cognition, or emotion, will be complete without understanding the development and function of zones. To this end, a special issue of this journal is dedicated to zones, and the purpose of this article is to summarize the research and review articles that are contained within. The special issue begins by considering some of the very first studies in the 1960s and 1970s that led to our modern understanding of this unique and defining anatomical substructure. Then, it considers the molecular analogs of longitudinal zones in the form of stripes in the cerebellar cortex and related sub-areas in the deep cerebellar nuclei, and it includes studies on the genetic underpinnings of stripes and zones. Several articles address the evolution of both embryonic clusters and adult zones across vertebrate species, and others discuss the functional and clinical relevance of zones. While we do not yet fully understand the role of zones with respect to motor behavior in all of its complexities, cerebellar function is clearly modular, and combinatorial models of complex motor movements based on multi-purpose modules are beginning to emerge. This special issue, by refocusing attention on this fundamental organization of the cerebellum, sets the stage for future studies that will more fully reveal the cellular, developmental, behavioral, and clinical relevance of zones.

Cell death as a regulator of cerebellar histogenesis and compartmentation.

Programmed cell death is essential for the homeostasis of tissues and organs. During the development of the central nervous system, programmed cell death is highly regulated and restricted to distinct developmental time points of histogenesis. In this review, we will summarize recent data on the temporal and spatial distribution of programmed Purkinje cell death within the cerebellar cortex. We point out that programmed cell death within distinct regions of the developing cerebellar cortex differs by type and its cellular consequences. We submit the concept that local Purkinje cell death is important for late compartmentation of the cerebellar cortex and its wiring. To support this hypothesis, we provide new data obtained from a cerebellar mutant with prolonged expression of Engrailed-2 specifically in cerebellar Purkinje cells which shows increased local physiological cell death and concomitant changes in the pattern of afferent innervation.

Sensorimotor enhancement in mouse mutants lacking the Purkinje cell-specific Gi/o modulator, Pcp2(L7).

Pcp2(L7) is a GoLoco domain protein specifically and abundantly expressed in cerebellar Purkinje cells. It has been hypothesized to "tune" G(i/o)-coupled receptor modulation of physiological effectors, including the P-type Ca(2+) channel. We have analyzed a mouse mutant in which the Pcp2(L7) gene was inactivated and find significant anatomical, behavioral and electrophysiological changes. Anatomically, we observed mild cerebellar hypoplasia. Behaviorally, the mutants were altered in modalities atypical for a traditional cerebellar mutant, and oddly, all of these changes could be considered functional enhancements. This includes increased asymptotic performance in gross motor learning, increased rate of acquisition in tone-conditioned fear, and enhanced pre-pulse inhibition of the acoustic startle response. Electrophysiological analysis of Purkinje cells in the mutants reveals depression of the complex spike waveform that may underlie the behavioral changes. Based on these observations we suggest that the Pcp2(L7) protein acts as a sensorimotor damper that modulates time- and sense-dependent changes in motor responses.

Pharmacological Prevention of Neonatal Opioid Withdrawal in a Pregnant Guinea Pig Model.

Newborns exposed to prenatal opioids often experience intense postnatal withdrawal after cessation of the opioid, called neonatal opioid withdrawal syndrome (NOWS), with limited pre- and postnatal therapeutic options available. In a prior study in pregnant mice we demonstrated that the peripherally selective opioid antagonist, 6ß-naltrexol (6BN), is a promising drug candidate for preventive prenatal treatment of NOWS, and a therapeutic mechanism was proposed based on preferential delivery of 6BN to fetal brain with relative exclusion from maternal brain. Here, we have developed methadone (MTD) treated pregnant guinea pigs as a physiologically more suitable model, enabling detection of robust spontaneous neonatal withdrawal. Prenatal MTD significantly aggravates two classic maternal separation stress behaviors in newborn guinea pigs: calling (vocalizing) and searching (locomotion) - natural attachment behaviors thought to be controlled by the endogenous opioid system. In addition, prenatal MTD significantly increases the levels of plasma cortisol in newborns, showing that cessation of MTD at birth engages the hypothalamic-pituitary-adrenal (HPA) axis. We find that co-administration of 6BN with MTD prevents these withdrawal symptoms in newborn pups with extreme potency (ID50 ∼0.02 mg/kg), at doses unlikely to induce maternal or fetal withdrawal or to interfere with opioid antinociception based on many prior studies in rodents and non-human primates. Furthermore, we demonstrate a similarly high potency of 6BN in preventing opioid withdrawal in adult guinea pigs (ID50 = 0.01 mg/kg). This high potency appears to run counter to our pharmacokinetic studies showing slow 6BN transit of both the placenta and maternal blood brain barrier in guinea pigs, and calls into question the preferential delivery mechanism. Rather, it suggests a novel receptor mechanism to account for the selectively high potency of 6BN to suppress opioid dependence at all developmental stages, even in adults, as compared to its well-established low potency as a classical opioid antagonist. In conclusion, 6BN is an attractive compound for development of a preventive therapy for NOWS.

The treasury of the commons: making use of public gene expression resources to better characterize the molecular diversity of inhibitory interneurons in the cerebellar cortex.

We mined the Allen Mouse Brain Atlas for genes expressed in cerebellar cortical inhibitory interneurons that would allow identification and possibly distinction of these cells. We identified some 90 genes that are highly expressed in specific subsets of cerebellar cortical inhibitory interneurons or various combinations thereof. Four genes are exclusively expressed, within the cerebellar cortex, in molecular layer interneurons, and ten genes label exclusively inhibitory interneurons in the granule cell layer or subsets thereof. Differential expression of many of these genes in cells residing in the lower versus the upper molecular layer provides evidence that these cells, traditionally referred to as basket and stellate cells, are indeed molecularly distinct. Two genes could be identified as novel markers for unipolar brush cells. Intersection of these data with embryonic expression patterns as documented in the genepaint repository does not support a hierarchical model of cerebellar interneuron development, but may be more easily reconciled with the view that cerebellar inhibitory interneurons derive from a common precursor pool from which they are specified only late into their development. The novel markers identified here should prove useful for probing the timing and mechanisms supporting cerebellar cortical interneuron specification and diversification.

Engrailed-2 regulates genes related to vesicle formation and transport in cerebellar Purkinje cells.

Engrailed transcription factors regulate survival, cell fate decisions and axon pathfinding in central neurons. En-2 can also attenuate Purkinje cell (PC) maturation. Here, we use array analysis to scrutinize gene expression in developing PCs overexpressing Engrailed-2 (L7En-2). The majority (70%) of regulated genes was found down-regulated in L7En-2 cerebella, consistent with the known repressive function of Engrailed-2. Differential gene expression, verified by in situ hybridization or Western blotting, was particularly evident during the first postnatal week, when L7En-2 PCs display conspicuous deficits in dendritogenesis. Functional classification revealed clusters of genes linked to vesicle formation and transport. Consistently, Golgi stacks located at the axonal pole of wild type PC somata were rarely detected in L7En-2 PCs. In addition, long continuous stretches of endoplasmic reticulum typically found around the axonal pole of wild type PCs were less frequently observed in transgenic cells. Engrailed-2 might therefore orchestrate PC survival and process formation as a regulator of subcellular organization.

3'UTR-dependent localization of a Purkinje cell messenger RNA in dendrites.

Pcp2(L7) is a Purkinje cell-specific GoLoco domain protein that modulates activation of Galphai/o proteins by G protein-coupled receptors. A likely downstream effector of this pathway is the P-type Ca(2+) channel, and thereby, the intrinsic electrophysiology of Purkinje cells could be modulated by Pcp2(L7). It has long been known that the Pcp2(L7) mRNA is abundantly localized in dendrites, suggesting the possibility of distal synthesis and local changes in levels of the protein. As a first step to uncover the trafficking and translational mechanisms for this mRNA, we have begun identifying the cis-acting sequences important for its localization in dendrites. Using expression of modified transgenes in vivo, we show that the 3'UTR, only 65 bases long, is necessary in this process.

Developmental expression and differentiation-related neuron-specific splicing of metastasis suppressor 1 (Mtss1) in normal and transformed cerebellar cells.

BACKGROUND: Mtss1 encodes an actin-binding protein, dysregulated in a variety of tumors, that interacts with sonic hedgehog/Gli signaling in epidermal cells. Given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of Mtss1 in the developing murine cerebellum and human medulloblastoma specimens. RESULTS: During development, Mtss1 is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. It is also expressed by granule cell precursor-derived medulloblastomas. In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 suggests that differential exon usage may affect interaction with Fyn and Src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. Further, this approach led to the identification of two evolutionary conserved nuclear localization sequences. These overlap with the actin filament binding site of Mtss1, and one also harbors a potential PKA and PKC phosphorylation site. CONCLUSION: Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.

Optical imaging of long-term depression in the mouse cerebellar cortex in vivo.

Conjunctive stimulation of climbing fiber and parallel fiber inputs results in long-term depression (LTD) at parallel fiber-Purkinje cell synapses. Although hypothesized to play a major role in cerebellar motor learning, there has been no characterization of the cellular and molecular mechanisms of LTD in the whole animal, let alone its spatial properties, both of which are critical to understanding the role of LTD in cerebellar function. Neutral red optical imaging of the cerebellar cortex in the anesthetized mouse was used to visualize the spatial patterns of activation. Stimulation of the parallel fibers evoked a transverse beam of optical activity, and stimulation of the contralateral inferior olive evoked parasagittal bands. Conjunctive stimulation of parallel fibers and climbing fibers induced a long-term decrease (at least 1 hr) in the optical response to subsequent parallel fiber activation confined to the region of interaction between these two inputs. Activation of climbing fibers alone failed to induce the long-term decrease. Field potential recordings confirmed that the depression is postsynaptic and restricted to the interaction site. The long-term depression in the beam was prevented by a group 1 metabotropic glutamate receptor (mGluR(1)) antagonist and was absent in transgenic mice selectively expressing an inhibitor of protein kinase C (PKC) in Purkinje cells. Conversely, the long-term depression occurred in the mGluR(4) knock-out mouse, consistent with its postsynaptic origin. In addition to providing the first visualization of parallel fiber-Purkinje cell LTD in the cerebellar cortex, this study demonstrates the spatial specificity of LTD and its dependence on mGluR(1) and PKC in vivo.

Engrailed-2 negatively regulates the onset of perinatal Purkinje cell differentiation.

The transcription factor Engrailed-2 is expressed in cerebellar Purkinje cells (PCs) throughout embryonic development but is downregulated in PCs after birth. Since the onset of PC differentiation coincides with this change of gene expression, we asked whether downregulation of Engrailed-2 is necessary for proper timing of PC differentiation. To investigate this, we used an L7En-2 transgenic mouse model in which Engrailed-2 expression in PCs is maintained beyond the day of birth. In these L7En-2 mice the onset of parvalbumin expression was delayed in all PCs by about 3 days; the spatial expression pattern, however, remained comparable to wildtype cerebella. Furthermore, parvalbumin expression resembled the known pattern of normal PC maturation, suggesting a direct link between parvalbumin expression and PC differentiation. Consistent with a delay of PC differentiation, we found that PCs of L7En-2 cerebella displayed a reduced tendency to align in the typical monolayer. The average size of L7En-2 PCs was reduced and the dendritic arbor developed more slowly than in wildtype PCs. In contrast, major morphological features of PCs were comparable in L7En-2 and wildtype cerebella after postnatal day 11. In addition, we observed a transient reduction of PC survival in organotypic slice cultures of L7En-2 cerebella in comparison with wildtype slice cultures. Since PC survival parallels PC differentiation in vitro, we propose that the observed delay in PC differentiation upon Engrailed-2 overexpression is an intrinsic property of Engrailed-2 activity, and that downregulation of Engrailed-2 in wildtype PCs around the day of birth is critical for the timing of distinct steps of PC differentiation.

Conservation of the developmentally regulated dendritic localization of a Purkinje cell-specific mRNA that encodes a G-protein modulator: comparison of rodent and human Pcp2(L7) gene structure and expression.

L7/Pcp-2 is a GoLoco domain protein that modulates the activation of Galpha(i) and Galpha(o). We have previously described the Purkinje cell-specific expression of the Pcp-2(L7) gene and the abundant localization of its mRNA in mouse cerebellar Purkinje cell dendrites. Here we report on two alternative cerebellar forms of the L7/Pcp-2 mRNA and protein by examination of the gene structures and cDNA sequences of the mouse, rat, and human genes. The structures of the rodent and human genes are very similar with the most notable difference in the genomic configuration of the first exon. Despite this difference, the human and rodent genes both encode two alternative mRNAs due to the choice of two transcriptional start positions. The two mRNA forms, in turn, predict two forms of the L7/Pcp-2 protein, which are both highly conserved across species. These two protein forms differ with respect to the number of GoLoco domains. Lastly we examined the issue of mRNA localization in dendrites. In mouse both mRNA forms are detectable in dendrites but their relative proportions change during development. In addition we performed in situ hybridization on a developmental series of human cerebellar sections and demonstrate that the L7/Pcp-2 mRNA is also localized in dendrites of humans. As previously described in the mouse the dendritic localization in humans is developmentally regulated being most prominent during the peak phase of synaptogenesis and decreasing dramatically with age. The conservation of all of these properties of both the L7/Pcp-2 protein and mRNA highlights their likely importance in controlling the development and/or motor control function of Purkinje cells.

A Purkinje cell specific GoLoco domain protein, L7/Pcp-2, modulates receptor-mediated inhibition of Cav2.1 Ca2+ channels in a dose-dependent manner.

L7/Pcp-2 is a GoLoco domain protein encoded by a Purkinje cell dendritic mRNA. Although biochemical interactions of GoLoco proteins with Galpha(o) and Galpha(i) are well documented, little is known about effector function modulation resulting from these interactions. The P-type Ca2+ channels might be physiological effectors of L7 because (1) they are the major voltage-dependent Ca2+ channels (VDCC) that modulate Purkinje cell output and (2) they are regulated by G(i/o) proteins. As a first step towards validating this hypothesis and to further understand the possible physiological effect of L7 protein and its two isoforms, we have coexpressed Ca(v)2.1 channels and kappa-opioid receptors (KORs) with varying amounts of L7A or L7B in Xenopus oocytes and measured ionic currents by two-electrode voltage clamping. Without receptor activation L7 did not alter the Ca2+ channel activity. With tonic and weak activation of the receptors, however, the Ca2+ channels were inhibited by 40-50%. This inhibition was enhanced by low, but dampened by high, expression levels of L7A and L7B and differences were observed between the two isoforms. The enhancing effect of L7 was occluded by overexpression of Gbetagamma, whereas the disinhibition was antagonized by overexpression of Galpha(o). We propose that L7 differentially affects the Galpha and Gbetagamma arms of receptor-induced G(i/o) signaling in a concentration-dependent manner, through which it increases the dynamic range of regulation of P/Q-type Ca2+ channels by G(i/o) protein-coupled receptors. This provides a framework for designing further experiments to determine how dendritic local fluctuations in L7 protein levels might influence signal processing in Purkinje cells.