A cyclic AMP related gene network in microglia is inversely regulated by morphine tolerance and withdrawal
Biological Psychiatry Global Open Science
Coffey, K;Lesiak, A;Marx, R;Vo, E;Garden, G;Neumaier, J;
| DOI: 10.1016/j.bpsgos.2021.07.011
Background Microglia have recently been implicated in opioid dependence and withdrawal. Mu Opioid (MOR) receptors are expressed in microglia, and microglia form intimate connections with nearby neurons. Accordingly, opioids have both direct (MOR mediated) and indirect (neuron-interaction mediated) effects on microglia function. Methods To investigate this directly, we used RNA sequencing of ribosome-associated RNAs from striatal microglia (RiboTag-Seq) after the induction of morphine tolerance and followed by naloxone precipitated withdrawal (n=16). We validated the RNA-Seq data by combining fluorescent in-situ hybridization with immunohistochemistry for microglia (n=18). Finally, we expressed and activated the Gi/o-coupled hM4Di DREADD receptor in CX3CR1-expressing cells during morphine withdrawal (n=18). Results We detected large, inverse changes in RNA translation following opioid tolerance and withdrawal. WGCNA analysis revealed an intriguing network of cAMP-associated genes that are known to be involved in microglial motility, morphology, and interactions with neurons that were downregulated with morphine tolerance and upregulated rapidly by withdrawal. Three-dimensional histological reconstruction of microglia allowed for volumetric, visual colocalization of mRNA within individual microglia that validated our bioinformatics results. Direct activation of Gi/o-coupled DREADD receptors in CX3CR1-expressing cells exacerbated signs of opioid withdrawal rather than mimicking the effects of morphine. Conclusions These results indicate that Gi-signaling and cAMP-associated gene networks are inversely engaged during opioid tolerance and early withdrawal, perhaps revealing a role of microglia in mitigating the consequences of opioids.
Al-Hasani R, McCall JG, Shin G, Gomez AM, Schmitz GP, Bernardi JM, Pyo CO, Park SI, Marcinkiewcz CM, Crowley NA, Krashes MJ, Lowell BB, Kash TL, Rogers JA, Bruchas MR.
PMID: 26335648 | DOI: 10.1016/j.neuron.2015.08.019
The nucleus accumbens (NAc) and the dynorphinergic system are widely implicated in motivated behaviors. Prior studies have shown that activation of the dynorphin-kappa opioid receptor (KOR) system leads to aversive, dysphoria-like behavior. However, the endogenous sources of dynorphin in these circuits remain unknown. We investigated whether dynorphinergic neuronal firing in the NAc is sufficient to induce aversive behaviors. We found that photostimulation of dynorphinergic cells in the ventral NAc shell elicits robust conditioned and real-time aversive behavior via KOR activation, and in contrast, photostimulation of dorsal NAc shell dynorphin cells induced a KOR-mediated place preference and was positively reinforcing. These results show previously unknown discrete subregions of dynorphin-containing cells in the NAc shell that selectively drive opposing behaviors. Understanding the discrete regional specificity by which NAc dynorphinerigic cells regulate preference and aversion provides insight into motivated behaviors that are dysregulated in stress, reward, and psychiatric disease.
Zhang L, Hernández VS, Vázquez-Juárez E, Chay FK, Barrio RA.
PMID: 27065810 | DOI: 10.3389/fncir.2016.00013
Water-homeostasis is a fundamental physiological process for terrestrial life. In vertebrates, thirst drives water intake, but the neuronal circuits that connect the physiology of water regulation with emotional context are poorly understood. Vasopressin (VP) is a prominent messenger in this circuit, as well as L-glutamate. We have investigated the role of a VP circuit and interaction between thirst and motivational behaviors evoked by life-threatening stimuli in rats. We demonstrate a direct pathway from hypothalamic paraventricular VP-expressing, glutamatergic magnocellular neurons to the medial division of lateral habenula (LHbM), a region containing GABAergic neurons. In vivo recording and juxtacellular labeling revealed that GABAergic neurons in the LHbM had locally branching axons, and received VP-positive axon terminal contacts on their dendrites. Water deprivation significantly reduced freezing and immobility behaviors evoked by innate fear and behavioral despair, respectively, accompanied by decreased Fos expression in the lateral habenula. Our results reveal a novel VP-expressing hypothalamus to the LHbM circuit that is likely to evoke GABA-mediated inhibition in the LHbM, which promotes escape behavior during stress coping.
Raam T, McAvoy KM, Besnard A, Veenema A, Sahay A.
PMID: 29222469 | DOI: 10.1038/s41467-017-02173-0
Oxytocin receptor (Oxtr) signaling in neural circuits mediating discrimination of social stimuli and affiliation or avoidance behavior is thought to guide social recognition. Remarkably, the physiological functions of Oxtrs in the hippocampus are not known. Here we demonstrate using genetic and pharmacological approaches that Oxtrs in the anterior dentate gyrus (aDG) and anterior CA2/CA3 (aCA2/CA3) of mice are necessary for discrimination of social, but not non-social, stimuli. Further, Oxtrs in aCA2/CA3 neurons recruit a population-based coding mechanism to mediate social stimuli discrimination. Optogenetic terminal-specific attenuation revealed a critical role for aCA2/CA3 outputs to posterior CA1 for discrimination of social stimuli. In contrast, aCA2/CA3 projections to aCA1 mediate discrimination of non-social stimuli. These studies identify a role for an aDG-CA2/CA3 axis of Oxtr expressing cells in discrimination of social stimuli and delineate a pathway relaying social memory computations in the anterior hippocampus to the posterior hippocampus to guide social recognition.
"Boldog E, Bakken TE, Hodge RD, Novotny M, Aevermann BD, Baka J, Bordé S, Close JL, Diez-Fuertes F, Ding SL, Faragó N, Kocsis AK, Kovács B, Maltzer Z, McCorrison JM, Miller JA, Molnár G, Oláh G, Ozsvár A, Rózsa M, Shehata SI, Smith KA, Sunkin SM, Tran D
PMID: 30150662 | DOI: 10.1038/s41593-018-0205-2
We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.
Nat Neurosci. 2019 Jan;22(1):47-56.
Fu H, Possenti A, Freer R, Nakano Y, Villegas NCH, Tang M, Cauhy PVM, Lassus BA, Chen S, Fowler SL, Figueroa HY, Huey ED, Johnson GVW, Vendruscolo M, Duff KE.
PMID: 30559469 | DOI: 10.1038/s41593-018-0298-7
Excitatory neurons are preferentially impaired in early Alzheimer's disease but the pathways contributing to their relative vulnerability remain largely unknown. Here we report that pathological tau accumulation takes place predominantly in excitatory neurons compared to inhibitory neurons, not only in the entorhinal cortex, a brain region affected in early Alzheimer's disease, but also in areas affected later by the disease. By analyzing RNA transcripts from single-nucleus RNA datasets, we identified a specific tau homeostasis signature of genes differentially expressed in excitatory compared to inhibitory neurons. One of the genes, BCL2-associated athanogene 3 (BAG3), a facilitator of autophagy, was identified as a hub, or master regulator, gene. We verified that reducing BAG3 levels in primary neurons exacerbated pathological tau accumulation, whereas BAG3 overexpression attenuated it. These results define a tau homeostasis signature that underlies the cellular and regional vulnerability of excitatory neurons to tau pathology.
Folorunso, OO;Brown, SE;Baruah, J;Harvey, TL;Jami, SA;Radzishevsky, I;Wolosker, H;McNally, JM;Gray, JA;Vasudevan, A;Balu, DT;
PMID: 37311798 | DOI: 10.1038/s41598-023-35615-5
The proper development and function of telencephalic GABAergic interneurons is critical for maintaining the excitation and inhibition (E/I) balance in cortical circuits. Glutamate contributes to cortical interneuron (CIN) development via N-methyl-D-aspartate receptors (NMDARs). NMDAR activation requires the binding of a co-agonist, either glycine or D-serine. D-serine (co-agonist at many mature forebrain synapses) is racemized by the neuronal enzyme serine racemase (SR) from L-serine. We utilized constitutive SR knockout (SR-/-) mice to investigate the effect of D-serine availability on the development of CINs and inhibitory synapses in the prelimbic cortex (PrL). We found that most immature Lhx6 + CINs expressed SR and the obligatory NMDAR subunit NR1. At embryonic day 15, SR-/- mice had an accumulation of GABA and increased mitotic proliferation in the ganglionic eminence and fewer Gad1 + (glutamic acid decarboxylase 67 kDa; GAD67) cells in the E18 neocortex. Lhx6 + cells develop into parvalbumin (PV+) and somatostatin (Sst+) CINs. In the PrL of postnatal day (PND) 16 SR-/- mice, there was a significant decrease in GAD67+ and PV+, but not SST + CIN density, which was associated with reduced inhibitory postsynaptic potentials in layer 2/3 pyramidal neurons. These results demonstrate that D-serine availability is essential for prenatal CIN development and postnatal cortical circuit maturation.
Brain Struct Funct. 2014 Nov 27.
de Kloet AD, Wang L, Ludin JA, Smith JA, Pioquinto DJ, Hiller H, Steckelings UM, Scheuer DA, Sumners C, Krause EG.
PMID: 25427952
Angiotensin-II acts at its type-1 receptor (AT1R) in the brain to regulate body fluid homeostasis, sympathetic outflow and blood pressure. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of limited ability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual immunohistochemistry (IHC)/ISH studies conducted in AT2R-eGFP reporter mice found that eGFP and AT2R mRNA were highly co-localized within the brain. Qualitative analysis of eGFP immunoreactivity in the brain then revealed localization to neurons within nuclei that regulate blood pressure, metabolism, and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]), as well as limbic and cortical areas known to impact stress responding and mood. Subsequently, dual IHC/ISH studies uncovered the phenotype of specific populations of AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-1 (80.3 ± 2.8 %), while a smaller subset express vesicular glutamate transporter-2 (18.2 ± 2.9 %) or AT1R (8.7 ± 1.0 %). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular nucleus (PVN) of the hypothalamus, eGFP immunoreactivity is localized to efferents terminating in the PVN and within GABAergic neurons surrounding this nucleus. These studies demonstrate that central AT2R are positioned to regulate blood pressure, metabolism, and stress responses.
Kim, JS;Williams, KC;Kirkland, RA;Schade, R;Freeman, KG;Cawthon, CR;Rautmann, AW;Smith, JM;Edwards, GL;Glenn, TC;Holmes, PV;de Lartigue, G;de La Serre, CB;
PMID: 37380023 | DOI: 10.1016/j.molmet.2023.101764
Obesity is associated with deficits in reward which have been linked to compensatory overeating. The vagus nerve is a direct neural pathway that conveys post-ingestive feedback from the gut to the brain, including the reward regions, and vagal activation causes stereotypical reward behaviors. Chronic high fat (HF) feeding alters vagal signaling potentially dampening food-associated reward. Microbiota composition changes rapidly with HF feeding, and a HF-type microbiota is sufficient to alter vagal structure and function. However, whether microbiota-driven alterations in vagal signaling affect host appetitive feeding behavior is unknown. Here, we investigate if microbiota composition modulates reward signaling and assess the role of the vagus in mediating microbiota to brain communication. Male germ-free Fisher rats were colonized with gastrointestinal contents from chow (low fat (LF) ConvLF) or HF (ConvHF) fed rats. Following colonization, ConvHF rats consumed significantly more food than ConvLF animals. ConvHF rats displayed lower feeding-induced extracellular DOPAC levels (a metabolite of dopamine) in the Nucleus Accumbens (NAc) as well as reduced motivation for HF foods compared to ConvLF rats. Dopamine receptor 2 (DDR2) expression levels in the NAc were also significantly lower in ConvHF animals. Similar deficits were observed in conventionally raised HF fed rats, showing that diet-driven alteration in reward can be initiated via microbiota. Selective gut to brain deafferentation restored DOPAC levels, DRD2 expression, and motivational drive in ConvHF rats. We concluded from these data that a HF-type microbiota is sufficient to alter appetitive feeding behavior and that bacteria to reward communication is mediated by the vagus nerve.
Moreno, E;Casajuana-Martin, N;Coyle, M;Campos, BC;Galaj, E;Del Torrent, CL;Seyedian, A;Rea, W;Cai, NS;Bonifazi, A;Florán, B;Xi, ZX;Guitart, X;Casadó, V;Newman, AH;Bishop, C;Pardo, L;Ferré, S;
PMID: 36182040 | DOI: 10.1016/j.phrs.2022.106476
A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4-116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4-116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson's disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.
Caprioli D, Venniro M, Zhang M, Bossert JM, Warren BL, Hope BT, Shaham Y.
PMID: 27980115 | DOI: 10.1523/JNEUROSCI.3091-16.2016
We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we studied the role of dorsolateral and dorsomedial striatum (DLS, DMS) in this incubation.We trained rats to self-administer palatable food pellets (6 days, 6-h/d) and methamphetamine (12 days, 6-h/d). We then assessed relapse to methamphetamine seeking under extinction conditions after 1 and 21 abstinence days. Between tests, the rats underwent voluntary abstinence (using a discrete choice procedure between methamphetamine and food; 20 trials/day) for 19 days. We used in situ hybridization to measure co-labeling of the activity marker Fos with Drd1 and Drd2 in DMS and DLS after the tests. Based on the in situ hybridization co-labeling results, we tested the causal role of DMS D1- and D2-family receptors, and DMS neuronal ensembles in 'incubated' methamphetamine seeking, using selective dopamine receptor antagonists (SCH39166 or raclopride) and the Daun02 chemogenetic inactivation procedure, respectively.Methamphetamine seeking was higher after 21 days of voluntary abstinence than after 1 day (incubation of methamphetamine craving). The 'incubated' response was associated with increased Fos expression in DMS but not DLS; Fos was co-labeled with both Drd1 and Drd2 DMS injections of SCH39166 or raclopride selectively decreased methamphetamine seeking after 21 abstinence days. In Fos-lacZ transgenic rats, selective inactivation of relapse test-activated Fos neurons in DMS on abstinence day 18 decreased incubated methamphetamine seeking on day 21.Results demonstrate a role of DMS dopamine D1 and D2-receptors in incubation of methamphetamine craving after voluntary abstinence and that DMS neuronal ensembles mediate this incubation.
SIGNIFICANCE STATEMENT:
In human addicts, abstinence is often self-imposed and relapse can be triggered by exposure to drug-associated cues that induce drug craving. We recently developed a rat model of incubation of methamphetamine craving after choice-based voluntary abstinence. Here, we used classical pharmacology, in situ hybridization, immunohistochemistry, and the Daun02 inactivation procedure to demonstrate a critical role of dorsomedial striatum neuronal ensembles in this new form of incubation of drug craving.
Quina LA1, Walker A1, Morton G1, Han V1, Turner EE2,3
PMID: 32332079 | DOI: 10.1523/ENEURO.0527-19.2020
The lateral habenula (LHb) sends complex projections to several areas of the mesopontine tegmentum, the raphe, and the hypothalamus. However, few markers have been available to distinguish subsets of LHb neurons that may serve these pathways. In order to address this complexity, we examined the mouse and rat LHb for neurons that express the GABA biosynthesis enzymes glutamate decarboxylase 1 and 2 (GAD1, GAD2), and the vesicular GABA transporter (VGAT). The mouse LHb contains a population of neurons that express GAD2, while the rat LHb contains discrete populations of neurons that express GAD1 and VGAT. However, we could not detect single neurons in either species that co-express a GABA synthetic enzyme and VGAT, suggesting that these LHb neurons do not use GABA for conventional synaptic transmission. Instead, all of the neuronal types expressing a GABAergic marker in both species showed co-expression of the glutamate transporter VGluT2. Anterograde tract-tracing of the projections of GAD2-expressing LHb neurons in Gad2Cre mice, combined with retrograde tracing from selected downstream nuclei, show that LHb-GAD2 neurons project selectively to the midline structures in the mesopontine tegmentum, including the median raphe and nucleus incertus, and only sparsely innervate the hypothalamus, rostromedial tegmental nucleus, and ventral tegmental area. Postsynaptic recording of LHb-GAD2 neuronal input to tegmental neurons confirms that glutamate, not GABA, is the fast neurotransmitter in this circuit. Thus GAD2 expression can serve as a marker for functional studies of excitatory neurons serving specific LHb output pathways in mice.SIGNFICANCE STATEMENT The lateral habenula provides a major link between subcortical forebrain areas and the dopamine (DA) and serotonin (5HT) systems of the midbrain and pons, and it has been implicated in reward mechanisms and the regulation of mood states. Few markers have been available for the specific cell types and complex output pathways of the lateral habenula. Here we examined the expression of genes mediating GABAergic and glutamatergic transmission in the mouse and rat LHb, where no neurons in either species expressed a full complement of GABAergic markers, and all expressed the glutamatergic marker VGluT2. Consistent with this, in mice the LHb GAD2 neurons are excitatory and appear to use only glutamate for fast synaptic transmission.
