Research Findings - Basic Neuroscience Research
Temporally Precise In Vivo Control of Intracellular Signaling
In the study of complex mammalian behaviors, technological limitations have prevented spatiotemporally precise control over intracellular signaling processes. Here Dr. Deisseroth of Stanford University and his research team report the development of a versatile family of genetically encoded optical tools ('optoXRs') that leverage common structure-function relationships among G-protein-coupled receptors (GPCRs) to recruit and control, with high spatiotemporal precision, receptor-initiated biochemical signaling pathways. In particular, they have developed and characterized two optoXRs that selectively recruit distinct, targeted signaling pathways in response to light. The two optoXRs exerted opposing effects on spike firing in nucleus accumbens in vivo, and precisely timed optoXR photostimulation in nucleus accumbens by itself sufficed to drive conditioned place preference in freely moving mice. The optoXR approach allows testing of hypotheses regarding the causal impact of biochemical signaling in behaving mammals, in a targetable and temporally precise manner. Airan RD, Thompson KR, Fenno LE, Bernstein H, Deisseroth, K. Temporally precise in vivo control of intracellular signalling. Nature. 2009; March 18; 1-5. Epub ahead of print.
Intracerebral BDNF Administration Prevents Cocaine-induced Increases in Glutamate Release and Cocaine Self-administration
Glutamatergic neurons originating from the dorsomedial prefrontal cortex (dmPFC) and projecting to the nucleus accumbens core (NAc) form a critical component of the reward circuitry that underlies reinstatement to cocaine-seeking behavior. Brain-derived neurotrophic factor (BDNF) activity is important for synaptic plasticity, is expressed by and modulates PFC-NAc neurons, and by itself enhances glutamatergic transmission. Cocaine also enhances glutatmate release. BDNF infusion into the dmPFC attenuates reinstatement to cocaine-seeking behavior, as well as some cocaine-induced molecular adaptations within the NAc. In the present study, it is demonstrated that a single intra-dmPFC infusion of BDNF prevents cocaine-induced increases in extracellular glutamate levels within the NAc. These data suggest that intra-PFC BDNF attenuates reinstatement to cocaine-seeking behavior and it does so by normalizing cocaine-induced neuroadaptations that alter glutamate neurotransmission within the NAc. Berglind, WJ, Whitfield, TW, LaLumiere, RT, Kalivas, PW, McGinty, JF. A single intra-PFC infusion of BDNF prevents cocaine-induced alterations in extracellular glutamate within the nucleus accumbens. J Neurosci, 2009;29(12):3715-19.
A Sensitizing D-Amphetamine Regimen Induces Long-Lasting Spinophilin Protein Upregulation in the Rat Striatum and Limbic Forebrain
Morphologic studies show that chronic regimens of psychostimulants increase dendritic length, branching, and overall spine density in the rat striatum. These long-term structural alterations in spines of striatal neurons may underlie sensitization-related alterations that contribute to addiction. The present study examined psychostimulant-induced changes in the levels of spinophilin, a protein found abundantly in dendritic spines, in brain regions implicated in psychostimulant-induced neuroplasticity. Rats received an escalating (1-8 mg/kg ip) regimen of d-amphetamine (twice daily) for 5 weeks, were tested for locomotor sensitization, and euthanized 28 days later. This amphetamine dosing regimen induced a significant sensitization of locomotor activity in these rats. Western blotting and radioimmunocytochemistry showed that spinophilin protein was upregulated in the striatum of the amphetamine-treated rats. Additionally, radioimmunocyto-chemical analyis revealed that spinophilin was also increased in the septum, hippocampus, amygdala, and cingulate cortex, but was unchanged in sensorimotor cortices. Because it binds to F-actin and protein phosphatase-1, spinophilin has been proposed as a protein linking synaptic transmission to changes in spine morphology. Radioimmunocytochemistry for spinophilin provides a novel approach to identification of brain regions whose neurons undergo dendritic change after chronic exposure to drugs of abuse. Boikess SR, Marshall JF. A sensitizing D-amphetamine regimen induces long-lasting spinophilin protein upregulation in the rat striatum and limbic forebrain. European Journal of Neuroscience. 2008;28:2099-2107.
Nucleus Accumbens CREB is Essential for Nicotine-Induced Conditioned Reward
Dr. Marina Picciotto and her colleagues at Yale University have continued their studies on the adaptive changes in brain and behavior that accompany repeated exposure to nicotine, including projects aimed at a better understanding of the role of transcription factor cyclic AMP-response element binding protein, or CREB. This factor is thought to be important for new gene transcription and in the phosphorylated form (pCREB) to promote long-term changes in synaptic strength. Earlier studies have associated nucleus accumbens (NAc) CREB activity with the modulation of cocaine and morphine reward, and have also revealed that nicotine conditioned place preference (CPP) is associated with NAc CREB activation. The present study showed that nicotine context conditioning led to elevated pCREB levels in the NAc shell, but not in the core of mice following placement in a nicotine-paired chamber in the absence of nicotine. To test if CREB activity in the NAc shell contributed to cue-induced responses that precipitate nicotine-seeking, Dr. Picciotto and her colleagues used viral-mediated gene transfer of a dominant-negative CREB construct in the NAc shell of C57BL/6J mice and found that disruption of CREB activation before training blocked nicotine place preference across a range of doses. Dr. Picciotto interpreted these studies to indicate that the NAc shell is a brain region where CREB activity is essential for nicotine CPP. Brunzell DH, Mineur YS, Neve RL, Picciotto MR. Nucleus accumbens CREB activity is necessary for nicotine conditioned place preference. Neuropsychopharmacology. 2009; Feb 11 [E-pub ahead of print]
Authentic Rat Embryonic Stem Cell Lines Established
The creation of knockout and knockin mice using mouse embryonic stem cells since the early 1980s has had tremendous impact on biological research. Now, Dr. Austin Smith and his group at the University of Cambridge, supported by NIDA funding, have demonstrated the ability to generate germline competent rat ES cells that will permit the creation of rats deficient in a gene of interest. Because the rat is a more tractable organism for physiology and pharmacology and is widely used in models of psychiatric and addiction disorders, this breakthrough will enable researchers in the field to generate transgenic rat models for these studies, and significantly advance the field. In addition, the new knowledge gained in generating these authentic stem cell lines will have high impact on human stem cell research. This research paves the way to targeted genetic manipulation in this important biomedical model species and provides a new test bed for cell therapies based on pluripotent stem cells. Buehr M, Meek S, Ure J, McLay R, Silva J, Yang J, Hall J, Blair K, Ying QL, Smith A. Authentic embryonic stem cells isolated from rat blastocysts. Cell. 2008;135:1287-98.
Maternal High-Fat Diet and Fetal Programming: Increased Proliferation of Hypothalamic Peptide-Producing Neurons That Increase Risk for Overeating and Obesity
Recent studies in adult and weanling rats show that dietary fat, in close association with circulating lipids, can stimulate expression of hypothalamic peptides involved in controlling food intake and body weight. Dr. Leibowitz of the Rockefeller University and her research team examined the possibility that a fat-rich diet during pregnancy alters the development of these peptide systems in utero, producing neuronal changes in the offspring that persist postnatally in the absence of the diet and have long-term consequences. The offspring of dams on a high-fat diet (HFD) versus balanced diet (BD), from embryonic day 6 to postnatal day 15 (P15), showed increased expression of orexigenic peptides, galanin, enkephalin, and dynorphin, in the paraventricular nucleus and orexin and melanin-concentrating hormone in the perifornical lateral hypothalamus. The increased density of these peptide-expressing neurons, evident in newborn offspring as well as P15 offspring cross-fostered at birth to dams on the BD, led them to examine events that might be occurring in utero. During gestation, the HFD stimulated the proliferation of neuroepithelial and neuronal precursor cells of the embryonic hypothalamic third ventricle. It also stimulated the proliferation and differentiation of neurons and their migration toward hypothalamic areas where ultimately a greater proportion of the new neurons expressed the orexigenic peptides. This increase in neurogenesis, closely associated with a marked increase in lipids in the blood, may have a role in producing the long-term behavioral and physiological changes observed in offspring after weaning, including an increase in food intake, preference for fat, hyperlipidemia, and higher body weight. Chang GQ, Gaysinskaya V, Karatayev O, Leibowitz SF. Maternal high-fat diet and fetal programming: increased proliferation of hypothalamic peptide-producing neurons that increase risk for overeating and obesity. J Neurosci. 2008;28(46):12107-19.
Optical Deconstruction of Parkinsonian Neural Circuitry
Deep brain stimulation (DBS) is a therapeutic option for intractable neurological and psychiatric disorders, including Parkinson's disease and major depression. Because of the heterogeneity of brain tissues where electrodes are placed, it has been challenging to elucidate the relevant target cell types or underlying mechanisms of DBS. Dr. Deisseroth of Stanford University and his team employed optogenetics and solid-state optics to systematically drive or inhibit an array of distinct circuit elements in freely moving Parkinsonian rodents, and found that therapeutic effects within the subthalamic nucleus can be accounted for by direct selective stimulation of afferent axons projecting to this region. In addition to providing insight into DBS mechanisms, these results demonstrate an optical approach for dissection of disease circuitry, and define the technological toolbox needed for systematic deconstruction of disease circuits by selectively controlling individual components. Gradinaru V, Mogri M, Thompson KR, Henderson J, Deisseroth K. Optical deconstruction of Parkinsonian neural circuitry. Sciencexpress. 2009; March 19; 1-13. Epub.
Gabapentin Acts within the Locus Coeruleus to Alleviate Neuropathic Pain
Gabapentin recruits descending inhibition to produce analgesia after nerve injury, but whether this is a local action in the brainstem is not known. It was hypothesized that gabapentin activates noradrenergic neurons in the locus coeruleus (LC) by a local action. Male rats underwent L5-L6 spinal nerve ligation (SNL) and received drugs by intra-LC or systemic routes for behavior testing, immunohistochemistry in the LC, and microdialysis in the spinal dorsal horn. In other studies, brainstem slices from normal and SNL animals were used for immunohistochemistry. SNL increased phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB)-expressing nuclei bilaterally in the LC, and increased noradrenaline release in the spinal dorsal horn. Gabapentin, whether in isolated brainstem slices or in conscious or anesthetized animals, increased pCREB-expressing nuclei in the LC. The net increase in pCREB expression by gabapentin did not differ between normal and SNL conditions. This gabapentin-induced pCREB activation in LC neurons was abolished by an AMPA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Intra-LC-injected gabapentin reduced hypersensitivity in SNL rats in a dose-dependent manner. Both intra-LC coadministration of CNQX and intrathecal administration of the alpha2-adrenoceptor antagonist idazoxan blocked antihypersensitivity by intra-LC gabapentin. Intravenous gabapentin induced noradrenaline release in the spinal dorsal horn. The net amount of noradrenaline release by gabapentin is larger in SNL rats compared with the normal condition, although the percentage increases from the baseline were the same. These results suggest that gabapentin acts directly in the brainstem via a glutamate-dependent mechanism to stimulate descending inhibition to produce antihypersensiti-ity after peripheral nerve injury. Hayashida K, Obata H, Nakajima K, Eisenach JC. Gabapentin acts within the locus coeruleus to alleviate neuropathic pain. Anesthesiology. 2008;109:1077-84.
Differential Effects of Methylphenidate and Cocaine on Dendritic Spines and Delta-FosB in the Striatum
Methylphenidate marketed as Ritalin is one of the most frequently used prescriptions for treating attention deficit hyperactivity disorder (ADHD). Both methylphenidate and cocaine are psychostimulants and like cocaine, methylphenidate can be abused when injected intravenously. This has raised concerns about whether long term treatment of children diagnoses as ADHD are at risk of developing substance abuse disorder. However, meta-analysis of ADHD children treated with methylphenidate suggests that these children are protected from developing subsequent substance abuse. Studies in juvenile rats also suggest that treatment with methylphenidate does not increase drug seeking behavior when these rats become mature. This raises the question of whether the actions of methylphenidate are different from those of cocaine. Dr. Paul Greengard and his colleagues compared the effect of chronically administered methylphenidate for two weeks with chronically administered cocaine for two weeks on medium spiny neurons in the striatum of transgenic mice. The medium spiny neurons expressing the DRD1 or DRD2 dopamine receptors in these transgenic mice are tagged with a fluorescent tag that allows the neurons to be identified. DRD1 expressing neurons are medium spiny neurons that project from the striatum to the ventral tegmental area and DRD2 expressing neurons are medium spiny neurons that project from the striatum to pallidum. Dr. Greengard and his colleagues report that both chronic cocaine and chronic methylphenidate increase the number of dendritic spines and delta-FosB in medium spiny neurons but the patterns observed in three regions of the striatum (dorsal striatum, nucleus accumbens shell, and nucleus accumbens core) are different for methylphenidate and cocaine. Chronic cocaine but not methylphenidate increased the density of spines in the dorsal striatum of DRD1 and DRD2 expressing spiny neurons. In the shell but not the core of the accumben both cocaine and methylphenidate increased the density of short dendritic spines in DRD2 expressing medium spiny neurons. Both methylphenidate and cocaine increase the density of dendritic spines in the shell and core of the accumbens. In contrast to cocaine, which increases the number of delta-FosB positive staining DRD1 and DRD2 neurons, methylphenidate increased only the number of delta-FosB positive DRD1 medium spiny neurons in all regions of the striatum. These results suggest that methylphenidate and cocaine produce different neuronal adaptations. Further work is needed to determine whether these differences can explain the reason that treatment of children with methylphenidate appears to decrease the risk of developing substance abuse disorder. Kim Y, Teylan MA, Baron M, Sands A, Nairn AC, Greengard P. Methylphenidate-induced dendritic spine formation and DeltaFosB expression in nucleus accumbens. Proc Natl Acad Sci U S A. 2009;106(8):2915-20.
D2 Dopamine and Type 1 Cannabinoid Receptors Both Target Downstream cAMP/PKA Signaling to Effect Neuroplasticity (Long-Term Depression)
Short- and long-term synaptic depression (LTD) are forms of neuroplasticity that are mediated by endogenous cannabinoid (eCB) signaling) in many brain regions. Endocannabinoid regulation is in turn affected by D(2) dopamine receptors, which show cooperativity with group I metabotropic glutamate receptors (mGluRs), ultimately inducing eCB-mediated LTD of glutamatergic excitatory and GABAergic inhibitory (I-LTD) synaptic transmission. Because D(2) receptors and group I mGluR agonists can induce the release of eCBs, it was widely accepted that D(2) receptors contributed to neuroplasticity (LTD) by regulating the release of eCBs. This paper challenges this hypothesis by showing that D(2) receptor activation does not enhance CB(1) receptor activation. Instead, D(2) receptor activation facilitates I-LTD induction via direct inhibition of cAMP-dependent protein kinase A (PKA) signaling. The authors demonstrate that the cAMP/PKA signaling pathway is the downstream effector for CB(1) receptors and is required for eCB-mediated I-LTD induction. Importantly, these results suggest that D(2) receptors and CB(1) receptors target the same downstream effector cAMP/PKA signaling pathway resulting in neuroplasticity (I-LTD). Further, D(2) receptor activation facilitates eCB-mediated I-LTD in dopamine neurons without enhancing CB(1) receptor activation. Pan B, Hillard CJ, Liu QS. D2 dopamine receptor activation facilitates endocannabinoid-mediated long-term synaptic depression of GABAergic synaptic transmission in midbrain dopamine neurons via cAMP-protein kinase A signaling. J Neurosci. 2008;28(52):14018-30.
Structure-Activity Relationships for a Novel Series of Dopamine D2-like Receptor Ligands Based on N-Substituted 3-Aryl-8-azabicyclo[3,2,1]octan-ol
Dopamine receptors, specifically D2-like receptors (D2R), have received particular attention in psychostimulant abuse because their availability has been shown to be related to cocaine's pleasurable effects in both human and primate studies. Although antipsychotic medications targeting the D2-like receptors have been clinically utilized for over half a century, an understanding of the complex relationship between dopamine receptor subtype activity, clinical efficacy, and side effects remains incomplete. Hence, D2R are clearly involved in drug reinforcement and addiction, and D2R selective antagonists have recently shown efficacy in animal models of drug abuse without incapacitating motor side effects. Continued study of the D3-like receptors (D3R) using selective ligands has proven critical for the understanding of dopamine receptor related mechanisms, yet it remains unclear which subtypes are necessary and have the most potential to target in medication discovery. Given D2-like receptor antagonism has been implicated in the origin of cataleptic and other motor side effects, the narrow therapeutic window of non-selective D2R antagonists might be improved with a critical ratio of D2R/D3R selectivity. Thus the discovery of subtype selective ligands with which to determine specific roles of each of these receptor targets has been an imperative step toward the development of more effective medications for the treatment of schizophrenia, Parkinson's disease, obesity, and substance abuse. To provide tools with which to further explore the role of the dopamine receptor system, Dr. Paul and his colleagues developed high affinity D2R and D3R selective ligands by chemically modifying the lead chemical template, D2R-selective antagonist, by replacing piperidine with a tropane ring that reversed the selectivity as seen in the parent compound. Further exploration of both N-substituted and aryl ring-substituted analogs resulted in the discovery of several high affinity D2R/D3R ligands with 3-benzofurylmethyl-substituents, that induced high affinity not achieved in similarly N-substituted piperidine analogues and significantly (470-fold) improved D3R binding affinity compared to the parent ligand, L741,626. X-ray crystallographic data revealed a distinctive spatial arrangement of pharmacophoric elements in the piperidinol vs. tropine analogues, providing clues for the discovery in SAR at the D2 and D3 receptor subtypes. Paul NM, Taylor M, Kumar R, Deschamps J, Luedke R, Newman AH. Structure-activity relationships for a novel series of dopamine D2-like receptor ligands based on N-Substituted-3-Aryl-8-azabicyclo[3,2,1]octan-3-ol. Journal of Medicinal Chemistry. 2008;51:6095-6109.
Predicting Nicotine Dependence
The genetics of nicotine dependence (ND) have been elusive because no single factor is likely to explain a large proportion of the complex trait of dependence. Genome-wide association studies (GWAS) have been a powerful tool to help identify important genetic factors for ND, and subsequent replications have provided strong confirmation of the association between particular genetic variants and ND. Dr. Ramoni and his group have moved beyond individual single nucleotide polymorphism (SNP) associations to identify predictive models of the ND phenotype. Predictive modeling is a logical complement to the association-based approach because predictive measures are 1) amenable to translation into clinical practice where they can be used in risk communication and counseling, and 2) are able to address some of the challenges of the analysis and interpretation of genome-wide data. Dr. Ramoni and his colleagues used Bayesian networks, which are multivariate dependency models that account for simultaneous associations and interactions among multiple factors, to build the predictive models. Out of the original 73 SNPs identified by the Bierut et al. 2007 study, 60 were incorporated into the model, along with age and sex. SNP rs2836823 alone had the highest single predictive accuracy on fitted values of 54.4% (P=0.002). However, combined with the network of the other variables, the model achieved a predictive accuracy on fitted values of 75% (P<0.0001). This modeling approach is an iterative process of developing an understanding of the genetic basis of ND, in which weak associations are successively improved upon by generating hypotheses on the basis of subgroups and assessing them in new cohorts. As new information is uncovered, data can be added to the model to help define etiology driven definitions of ND and to reveal new targets for ND treatments. Ramoni RB, Saccone NL, Hatsukami DK, Bierut LJ, Ramoni MF. A testable prognositic model of nicotine dependence. J. Neurogenetics. 2009; Jan 31:1-10 [Epub ahead of print].
Morphine Leads to an Inhibition of CXCR4 Signaling in Neurons via a Ferritin Heavy Chain Mechanism
Dr. Olimpia Meucci and colleagues at Drexel University, Columbia University, and Washington University have been investigating the effects of μ-opioid receptor agonists on CXCR4 signaling in neurons, and the mechanisms involved in regulation of neuronal CXCR4 by opiates. CXCR4 is a CXCL12 chemokine receptor that promotes neuronal survival, plays a critical role in neuronal development, can be regulated by phosphorylation, and acts as a coreceptor for HIV envelope protein gp120. Opioids and chemokine systems can reciprocally influence the other's function, but the mechanism by which μ-opioid agonists modulate CXCR4 signaling is unknown. To that end, investigators tested whether opioid and chemokine receptors directly interact in neurons, using a glia-free neuronal culture system and treating with DAMGO (μ-opioid agonist) or morphine. They found that both drugs blocked CXCL12-induced phosphorylation in neurons and inhibited CXCL12-dependent signaling. They also tested whether in vivo morphine treatment alters CXCL12 responses and found that morphine transiently inhibited CXCL12-induced G-protein activation. Moreover, neither of the findings described above are due to a reduction in CXCR4 expression, as determined by RT-PCR and Westerns. The next set of experiments revealed that pretreatment with morphine completely abolished CXCR4 phosphorylation induced by CXCL12, suggesting that opioids may alter neuronal CXCR4 signaling by interfering with receptor activation, internalization, and recycling. Additional findings suggest that opioid treatment induced long-term adaptations in neurons that require de novo protein synthesis and resulted in deficits of CXCR4 signaling. To understand the mechanism underlying a decrease in CXCL12-induced phosphorylation, they sought to determine if Ferritin Heavy Chain (FHC), which has been shown to inhibit CXCR4 signaling, is increased by opioid exposure and thus inhibits receptor signaling. DAMGO and morphine caused a time-dependent increase in neuronal FHC levels, and an increase in FHC protein was also reported in the cortex of morphine-treated animals. The onset and decay of FHC upregulation inversely correlated with CXCR4 activation. These results point to a crucial role of FHC in mediating the effect of opioids on neuronal CXCR4. Lastly, in FHC-deficient cells, DAMGO pretreatment did not affect the phosphorylation induced by CXCL12. These data provide strong evidence that the upregulation of FHC levels is an important factor in the inhibition of CXCR4 signaling by opioids, and may be a potential mechanism by which opioids reduce the neuroprotective functions of CXCR4 and lead to increased susceptibility to infectious disorders. Sengupta R, Burbassi S, Shimizu S, Cappello S, Vallee RB, Rubin JB, Meucci O. Morphine increases brain levels of ferritin heavy chain leading to inhibition of CXCR4-mediated survival signaling in neurons. Journal of Neuroscience. 2009;29:2534-44.
Mechanism of Persistent Enhanced Synaptic Strength in Nucleus Accumbens
The nucleus accumbens (NAc) plays a central role in mediating motivated behaviors related to natural rewards and drugs of abuse. Ninety percent of the neurons in the NAc are "medium spiny" neurons (MSNs); MSNs project from the NAc, and are an interface from limbic to motor systems. MSNs receive inputs related to motivational state from dopamine (DA) neurons originating in the ventral mesencephalon and from glutamate neurons originating in the prefrontal cortex (PFC) and limbic regions such as the hippocampus and amygdala. Before we can understand the mechanisms underlying addiction, we must understand how the DA and glutamate inputs interact at MSNs. Marina Wolf's group tackled this problem by developing an in vitro system consisting of rat NAc neurons co-cultured with labeled PFC neurons obtained from enhanced cyan fluorescent protein-expressing mice. The cortical neurons fluoresce and provide excitatory input to NAc neurons and can be distinguished from the NAc neurons in culture. They first showed that brief DA D1 agonist exposure increased AMPA receptor (AMPAR) insertion onto extrasynaptic regions of MSN processes through a mechanism requiring protein kinase A. This facilitated the Ca2+/calmodulin dependent protein kinase II (CaMKII)-dependent synaptic incorporation of AMPAR in response to subsequent NMDA glutamate receptor (NMDAR) stimulation. Through this mechanism, DA may promote reward- and drug-related plasticity in the NAc. Then, to model effects of repeated in vivo cocaine exposure, they treated the co-cultures with DA on days 7, 9 and 11 in culture. On day 15, MSNs exhibited increased synaptic AMPAR levels. This required CaMKII activation during the 4-day "withdrawal" period. Further, D1 agonist exposure on day 15 no longer increased AMPAR surface expression. NMDAR surface expression was not altered by acute or repeated DA receptor stimulation. Since it is known that the ratio of AMPAR to NMDAR determines persistent synaptic strength, the present results suggest that psychomotor stimulants, by increasing DA levels, may initially facilitate plasticity in the NAc, perhaps contributing to learning of drug-seeking behaviors. After drug withdrawal, NAc MSNs may be more responsive to glutamate inputs that trigger drug seeking. Sun X, Milovanovic M, Zhao Y, Wolf ME. Acute and chronic dopamine receptor stimulation modulates AMPA receptor trafficking in nucleus accumbens neurons cocultured with prefrontal cortex neurons. J Neuroscience. 2008;28:4216-30.
Expression and Adhesion Profiles of SynCAM Molecules Indicate Distinct Neuronal Functions
Cell-cell interactions through adhesion molecules play key roles in the development of the nervous system. Synaptic cell adhesion molecules (SynCAMs) comprise a group of four immunoglobulin (Ig) superfamily members that mediate adhesion and are prominently expressed in the brain. Although SynCAMs have been implicated in the differentiation of neurons, there has been no comprehensive analysis of their expression patterns. Here Dr. Biederer of Yale University examines the spatiotemporal expression patterns of SynCAMs by using reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistological techniques. SynCAMs 1-4 are widely expressed throughout the developing and adult central nervous system and are present in both excitatory and inhibitory neurons. Each SynCAM has a distinct spatiotemporal expression pattern in all regions analyzed in developing and mature mouse brain and it is particularly notable in the cerebellum, where SynCAMs display highly distinct expression in cerebellar granule and Purkinje cells. These unique expression profiles are complemented by specific heterophilic adhesion patterns of SynCAM family members, as shown by cell overlay experiments. Three prominent interactions are observed, mediated by the extracellular domains of SynCAMs 1/2, 2/4, and 3/4. These expression and adhesion profiles of SynCAMs together with their previously reported functions in synapse organization indicate that SynCAM proteins contribute importantly to the synaptic circuitry of the central nervous system. Thomas LA, Akins MR, Biederer T. Expression and adhesion profiles of SynCAM molecules indicate distinct neuronal functions. J Comp Neurol. 2008;510(1):47-67.
CREB Regulation of Channel Gene Expression Underlies Rapid Drug Tolerance
Dr. Nigel Atkinson and co-workers exploit the genetically powerful fruit fly model system to investigate the molecular basis of inhalant tolerance. Previously, Dr. Atkinson has shown that a single exposure to inhalant can lead to epigenetic changes in the chromatin (the DNA/protein complex in the nucleus of a cell) near the "slowpoke" potassium channel gene, leading to altered expression of the slowpoke gene and reduced sensitivity (tolerance) to additional inhalant exposures. These studies are based on the observation that animals become tolerant to sedation by organic solvents, which can be abused as inhalants, and this reduced sensitivity to inhalant requires increased expression of the slowpoke potassium channel which in turn alters neuronal function. The epigenetic changes involved are believed to lead to a more "open" chromatin conformation, allowing transcription factors to bind to DNA elements more readily. Which transcription factors are important in this process? In this follow up study, Dr. Atkinson and co-workers investigate the role of the transcription factor CREB which has been previously linked to processes critical for addiction. Sedation with benzyl alcohol leads to increased expression of positively acting CREB isoforms and reduced expression of negatively acting CREB isoforms (including dCREB2). Specifically the dCREB2 isoform shows increased occupancy at the slowpoke promoter immediately after benzyl alcohol sedation in a chomatin immunopreciptation assay. Animals with a knockout in dCREB2 no longer have increased benzyl alcohol induced slowpoke gene expression and also no longer develop tolerance to this organic solvent. Overall this work provides insight into the precise mechanisms by which exposure to an inhalant can lead to gene expression changes of a single gene, resulting in altered neuronal function and altered behavioral responses of an animal to future inhalant exposure. Although this work investigated an inhalant, analogous mechanisms may be utilized for responses to other drugs of abuse. Wang Y, Ghezzi A, Yin JCP, Atkinson NS. CREB regulation of BK channel gene expression underlies rapid drug tolerance. Genes, Brain and Behavior. 2009; Feb 9. [Epub ahead of print].
Sister Neurons Prefer Sister Neurons: Preferential Synaptic Formation Between Sister Neurons in the Radial Column of the Developing Cortex
Cortical neurons are often organized into columns in function. In many situations a single neuron in a column almost selectively communicates only with neurons above or below the column, but not the adjacent neurons. Such functional organization is pivotal for critical cortical information processing, such as seen in ocular dominance columns. However, how this selective communication within a column is established in cortical development is not known. Dr. Song-Hai Shi, a NIDA supported neurobiologist at Memorial Sloan Kettering and Cornell University, reports in Nature that sister neurons derived from the same mother radial glial cell during cortical formation migrate radially into destined cortical layers in the same column, and preferentially form functional excitatory synapses between each other within the column. The research team labeled ontogenetic radial clones of excitatory neurons in the mouse neocortex by in utero intraventricular injection of enhanced green fluorescent protein (EGFP)-expressing retroviruses around the onset of the peak phase of neocortical neurogenesis. The columns of sister neurons are then identified by the EGFP expression. Multiple-electrode whole-cell recordings were performed to probe synapse formation among these EGFP-labeled sister excitatory neurons in radial clones and the adjacent non-siblings during postnatal stages. They found that radially aligned sister excitatory neurons have a propensity for developing unidirectional chemical synapses with each other rather than with neighboring non-siblings. Moreover, these synaptic connections display the same interlaminar directional preference as those observed in the mature neocortex. These results indicate that specific microcircuits develop preferentially within ontogenetic radial clones of excitatory neurons in the developing neocortex and contribute to the emergence of functional columnar microarchitectures in the mature neocortex. Yu YC, Bultje RS, Wang X, Shi SH. Specific synapses develop preferentially among sister excitatory neurons in the neocortex. Nature. 2009;458:501-5.
The G-protein Coupled Receptor, Neurokinin 1, Mediates Opioid-Induced Endocytosis and Desensitization of Mu-Opioid Receptors
Mu-Opioid receptors (MORs) are G-protein-coupled receptors (GPCR) that mediate the physiological effects of endogenous opioid neuropeptides and opiate drugs such as morphine. MORs are coexpressed with neurokinin 1 receptors (NK1Rs) in several regions of the CNS that control opioid dependence and reward, and NK1R activation itself affects opioid reward. However, how NK1Rs regulate the mu opiate system is unknown. These researchers report that ligand-induced activation of NK1Rs mediates an NK1R-dependent sequestration of arrestins on endosome membranes that results in the cell-autonomous and nonreciprocal inhibition of MOR endocytosis. NK1R-mediated regulation of MOR trafficking was associated with a reduction in the usual opioid-induced desensitization of adenylyl cyclase signaling in striatal neurons. Additionally, heterologous regulation of MOR trafficking was observed in both amygdala and locus coeruleus neurons that naturally coexpress these receptors. These results identify a cell-autonomous mechanism that may underlie the highly specific effects of NK1R on opioid signaling and suggest, more generally, that receptor-specific trafficking of arrestins may represent a fundamental mechanism for coordinating distinct GPCR-mediated signals at the level of individual CNS neurons. Yu YJ, Arttamangkul S, Evans CJ, Williams JT, von Zastrow M. Neurokinin 1 receptors regulate morphine-induced endocytosis and desensitization of mu-opioid receptors in CNS neurons. J Neurosci. 2009;29(1):222-33.
Pro-Opiomelanocortin Gene Variation Related to Alcohol or Drug Dependence:
Evidence and Replications Across Family- and Population-based Studies
Opioidergic neurotransmission is critical in many, possibly all, forms of substance dependence. Several opioid-system genes have been shown to be associated with substance dependence disorders. The pro-opiomelanocortin gene (POMC) encodes several peptides important for endogenous opioidergic neurotransmission. The investigators tested whether POMC genetic variation affects risk for substance dependence. Five noncoding single nucleotide polymorphisms spanning POMC were examined in independent family and case-control samples. Family-based studies included 854 subjects from 319 African American (AA) families and 761 subjects from 313 European American (EA) families. Each family had a pair of siblings affected with cocaine and/or opioid dependence. Case-control studies included 791 cases (455 AAs and 336 EAs) affected with alcohol, cocaine, and/or opioid dependence and 682 control subjects (199 AAs and 483 EAs). Family-based analyses revealed an association of rs6719226 with opioid dependence in AA families and rs6713532 with cocaine dependence in EA families (p = .010-.044). Case-control analyses demonstrated an association of rs6713532 with alcohol or cocaine dependence in EAs (p(allele-wise) = .003-.008). Moreover, the minor allele of rs1866146 was found to be a risk factor for cocaine or opioid dependence in AAs (p(allele-wise) = .010-.017) and for alcohol, cocaine, or opioid dependence in EAs (p(allele-wise) = .001-.003). Logistic regression analyses in which sex and age were considered and population stratification analyses confirmed these findings. Additionally, specific haplotypes increased risk for cocaine dependence (p = .023) in AAs and opioid dependence (p = .012) in EAs. Given these replicated results, the authors concluded that variation in POMC confers vulnerability to multiple forms of substance dependence. Zhang H, Kranzler HR, Weiss RD, Luo X, Brady KT, Anton RF, Farrer LA, Gelernter J. Pro-Opiomelanocortin gene variation related to alcohol or drug dependence: evidence and replications across family- and population-based studies. Biol Psychiatry. 2009; Feb 12 [Epub ahead of print].
Mu Opioid and Cholecystokinin Receptor Complexes
Dr. Philip Portoghese of the University of Minnesota and Dr. Laurence Miller of the Mayo Clinic in Arizona are investigating the physical association of the mu receptor (MOR) with the cholecystokinin receptor (CCK2) in the central nervous system. These two receptors have been shown to overlap in certain brain areas, particularly in medullary neurons shown to co-express the CCK2R and the MOR. By analogy with heterodimeric associations in the lipid cell membrane found for other GPCRs, such as MOR/DOR, MOR/CB1, KOR/DOR, this study attempts to show whether homodimers and heterodimers of CCK2 and MOR exist, and whether a bifunctional ligand containing a CCK2 antagonist pharmacophore linked to a MOR agonist could serve the dual purpose of treating pain, and reducing the tolerance developing with chronic administration of a MOR agonist. In this case, the MOR agonist oxymorphone was used as the MOR pharmacophore, and the CCK2 antagonist L-365,260 as the second pharmacophore, separated by a linker of 9, 16, 18, or 22 atoms. The basis of examination was the use of BRET technology (bioluminescence resonance energy transfer) in COS cells between recombinant receptors tagged either with luminescent Rluc (renilla luciferase enzyme, energy donor) or yellow fluorescent protein (YFP, energy acceptor), as well as equilibrium binding (radiolabeled CCK or DAMGO), calcium mobilization assay, and in-vivo measure of tolerance in the mouse tail-flick assay. In brief, the investigators found BRET signals as evidence of heteordimers of CCK2-MOR "induced" only in the presence of bivalent ligands with a spacer of 16, 18, or 22 atoms. Evidence for constitutive homodimers of CCK2 or MORs was also found, in the absence of any ligands, and the bivalent ligands did not affect these observed homodimers. Monovalent ligands containing only one of the two pharmacophores competed with the bivalent ligands (reducing the BRET signals) only in the case of the CCK2 ligand, but not the monovalent MOR ligand. Equilibrium binding Ki values measured in CHO cell membranes co-expressing MORs and CCK2s were comparable to the values found in cells expressing only the MOR receptor, but the binding to the CCK2 receptor was improved in the system expressing both receptors. Moderate intracellular calcium mobilization as a functional test was observed in CHO cells co-expressing the MOR and CCK2 receptors, for one bivalent ligand (18 atom spacer) and for the MOR monovalent ligand. Tolerance in the radiant heat tail-flick assay (in terms of effective dose) was not seen for any of the three bivalent ligands given to mice by the icv route. This paper suggests that use of bivalent ligands to observe heteromeric associations between different GPCRs continues to be a useful technique. Information about the effects of bivalent ligands on receptor dimeric conformations, their signaling pathways, and their therapeutic possibilities awaits further work. Zheng Y, Akgun E, Harikumar KG, Hopson J, Powers MD, Lunzer MM, Miller LJ, Portoghese PS. Induced association of mu opioid and cholecystokinin (CCK2) receptors by novel bivalent ligands. J Med Chem. 2009;52(2):247-58.