Philosophical transactions of the Royal Society of London. Series B, Biological sciences
Frehner, SS;Dooley, KT;Palumbo, MC;Smith, AL;Goodman, MM;Bales, KL;Freeman, SM;
PMID: 35858098 | DOI: 10.1098/rstb.2021.0118
Oxytocin is an endogenous neuropeptide hormone that influences social behaviour and bonding in mammals. Variations in oxytocin receptor (OXTR) expression may play a role in the social deficits seen in autism spectrum disorder. Previous studies from our laboratory found a dense population of OXTR in the human substantia nigra (SN), a basal ganglia structure in the midbrain that is important in both movement and reward pathways. Here, we explore whether differences in OXTR can be identified in the dopaminergic SN pars compacta of individuals with autism. Postmortem human brain tissue specimens were processed for OXTR autoradiography from four groups: males with autism, females with autism, typically developing (TD) males and TD females. We found that females with autism had significantly lower levels of OXTR than the other groups. To examine potential gene expression differences, we performed in situ hybridization in adjacent slides to visualize and quantify OXTR mRNA as well as mRNA for tyrosine hydroxylase. We found no differences in mRNA levels for either gene across the four groups. These results suggest that a dysregulation in local OXTR protein translation or increased OXTR internalization/recycling may contribute to the differences in social symptoms seen in females with autism. This article is part of the theme issue 'Interplays between oxytocin and other neuromodulators in shaping complex social behaviours'.
Skiteva, O;Yao, N;Mantas, I;Zhang, X;Perlmann, T;Svenningsson, P;Chergui, K;
PMID: 37029193 | DOI: 10.1038/s41531-023-00500-5
In Parkinson's disease (PD), axons of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) degenerate before their cell bodies. Calcium influx during pacemaker firing might contribute to neuronal loss, but it is not known if dysfunctions of voltage-gated calcium channels (VGCCs) occur in DA neurons somata and axon terminals. We investigated T-type and L-type VGCCs in SNc-DA neurons of two mouse models of PD: mice with a deletion of the Nurr1 gene in DA neurons from an adult age (cNurr1 mice), and mice bearing the G2019S mutation in the gene coding for LRRK2 (G2019S mice). Adult cNurr1 mice displayed motor and DA deficits, while middle-aged G2019S mice did not. The number and morphology of SNc-DA neurons, most of their intrinsic membrane properties and pacemaker firing were unaltered in cNurr1 and G2019S mice compared to their control and wild-type littermates. L-type VGCCs contributed to the pacemaker firing of SNc-DA neurons in G2019S mice, but not in control, wild-type, and cNurr1 mice. In cNurr1 mice, but not G2019S mice, the contribution of T-type VGCCs to the pacemaker firing of SNc-DA neurons was reduced, and somatic dopamine-D2 autoreceptors desensitized more. Altered contribution of L-type and T-type VGCCs to the pacemaker firing was not observed in the presence of a LRRK2 kinase inhibitor in G2019S mice, and in the presence of a flavonoid with antioxidant activity in G2019S and cNurr1 mice. The role of L-type and T-type VGCCs in controlling dopamine release from axon terminals in the striatum was unaltered in cNurr1 and G2019S mice. Our findings uncover opposite changes, linked to oxidative stress, in the function of two VGCCs in DA neurons somata, but not axon terminals, in two different experimental PD models.
Cline, MM;Juarez, B;Hunker, A;Regiarto, EG;Hariadi, B;Soden, ME;Zweifel, LS;
PMID: 36927614 | DOI: 10.7554/eLife.83760
The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding (Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, the reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Simultaneous loss of Ntn1 function in both cell types largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for Ntn1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.
Brain Struct Funct. 2015 Jul 10.
Hackett TA, Clause AR, Takahata T, Hackett NJ, Polley DB.
PMID: 26159773
Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11-P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1 + and VGluT2 + transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT + transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, perisomatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the observed changes in gene and protein expression, regional architecture, and morphology relate to-and to some extent may enable-the emergence of mature sound-evoked activity patterns. In that regard, the findings of this study expand our understanding of the presynaptic mechanisms that regulate critical period formation associated with experience-dependent refinement of sound processing in auditory forebrain circuits.
Godefroy, D;Boukhzar, L;Mallouki, BY;Carpentier, E;Dubessy, C;Chigr, F;Tillet, Y;Anouar, Y;
PMID: 35066506 | DOI: 10.1159/000522091
Selenoprotein T (SELENOT), a PACAP-regulated thioredoxin-like protein, plays a role in catecholamine secretion and protects dopaminergic neurons. However, the role of SELENOT in the establishment of the catecholaminergic (CA) neuronal system is not known yet.We analyzed by immunohistochemistry and RNAscope in situ hybridization the distribution of SELENOT and the expression of its mRNA, respectively. In addition, 3D imaging involving immunostaining in toto, clearing through the iDISCO+ method, acquisitions by light sheet microscopy and processing of 3D images was performed to map the CA neuronal system. A semi-automatic quantification of 3D images was carried out.SELENOT protein and mRNA are widely distributed in the mouse brain, with important local variations. Three-dimensional mapping, through tyrosine hydroxylase (TH) labeling, and semi-automated quantification of CA neurons in brain-specific SELENOT knockout mice showed a significant decrease in the number of TH-positive neurons in the area postrema (AP-A2), the A11 cell group (A11) and the zona incerta (ZI-A13) of SELENOT-deficient females, and in the hypothalamus (Hyp-A12-A14-A15) of SELENOT-deficient females and males.These results showed that SELENOT is diffusely expressed in the mouse brain and that its deficiency impacts CA neuron distribution in different brain areas including Hyp-A12-A14-A15, in both male and female mice.S. Karger AG, Basel.
Krajewski-Hall SJ, Miranda Dos Santos F, McMullen NT, Blackmore EM, Rance NE.
PMID: 30753503 | DOI: 10.1210/en.2018-00934
We have proposed that KNDy (kisspeptin/neurokinin B/dynorphin) neurons contribute to hot flushes via projections to neurokinin 3 receptor (NK3R) expressing neurons in the median preoptic nucleus (MnPO). To characterize the thermoregulatory role of MnPO NK3R neurons in female mice, we ablated these neurons using injections of saporin toxin conjugated to a selective NK3R agonist. Loss of MnPO NK3R neurons increased core temperature (TCORE) during the light phase, with frequency distributions indicating a regulated shift in the balance point. The rise in TCORE in ablated mice occurred despite changes in ambient temperature (TAMBIENT) and regardless of estrogen status. We next determined if an acute increase in TAMBIENT or higher TCORE would induce Fos in preoptic EGFP-immunoreactive neurons in Tacr3-EGFP mice. Fos-activation was increased in the MnPO, but there was no induction of Fos in NK3R (EGFP-immunoreactive) neurons. Thus, MnPO NK3R neurons are not activated by warm thermosensors in the skin or viscera and are not warm-sensitive neurons. Finally, RNAscope was used to determine if Tacr3 (NK3R) mRNA was co-expressed with VGLUT2 or VGAT mRNA, markers of glutamatergic or GABAergic neurotransmission, respectively. Interestingly, 94% of NK3R neurons in the MnPO were glutamatergic, whereas in the adjacent MPA, 97% of NK3R neurons were GABAergic. Thus, NK3R neurons in the MnPO are glutamatergic and play a role in reducing TCORE, but they are not activated by warm thermal stimuli (internal or external). These studies suggest that KNDy neurons modulate thermosensory pathways for heat-defense indirectly, via a subpopulation of glutamatergic MnPO neurons that express NK3R.
Hackett TA
PMID: 30315630 | DOI: 10.1002/ar.23907
In the brain, purines such as ATP and adenosine can function as neurotransmitters and co-transmitters, or serve as signals in neuron-glial interactions. In thalamocortical (TC) projections to sensory cortex, adenosine functions as a negative regulator of glutamate release via activation of the presynaptic adenosine A1 receptor (A1 R). In the auditory forebrain, restriction of A1 R-adenosine signaling in medial geniculate (MG) neurons is sufficient to extend LTP, LTD, and tonotopic map plasticity in adult mice for months beyond the critical period. Interfering with adenosine signaling in primary auditory cortex (A1) does not contribute to these forms of plasticity, suggesting regional differences in the roles of A1 R-mediated adenosine signaling in the forebrain. To advance understanding of the circuitry, in situ hybridization was used to localize neuronal and glial cell types in the auditory forebrain that express A1 R transcripts (Adora1), based on co-expression with cell-specific markers for neuronal and glial subtypes. In A1, Adora1 transcripts were concentrated in L3/4 and L6 of glutamatergic neurons. Subpopulations of GABAergic neurons, astrocytes, oligodendrocytes, and microglia expressed lower levels of Adora1. In MG, Adora1 was expressed by glutamatergic neurons in all divisions, and subpopulations of all glial classes. The collective findings imply that A1 R-mediated signaling broadly extends to all subdivisions of auditory cortex and MG. Selective expression by neuronal and glial subpopulations suggests that experimental manipulations of A1 R-adenosine signaling could impact several cell types, depending on their location. Strategies to target Adora1 in specific cell types can be developed from the data generated here.
Haidar M, Tin K, Zhang C, Nategh M, Covita J, Wykes AD, Rogers J and Gundlach AL
PMID: 30906254 | DOI: 10.3389/fnana.2019.00030
Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3 (loxP/loxP) mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV((1/2))-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.
Han X, He Y, Bi GH, Zhang HY, Song R, Liu QR, Egan JM, Gardner EL, Li J, Xi ZX.
PMID: 28951549 | DOI: 10.1038/s41598-017-12399-z
Cannabis can be rewarding or aversive. Cannabis reward is believed to be mediated by activation of cannabinoid CB1 receptors (CB1Rs) on GABAergic neurons that disinhibit dopaminergic neurons in the ventral tegmental area (VTA). However, little is known about the mechanisms underlying cannabis aversion in rodents. In the present study, CB1Rs are found not only on VTA GABAergic neurons, but also on VTA glutamatergic neurons that express vesicular glutamate transporter 2 (VgluT2). We then used Cre-Loxp transgenic technology to selectively delete CB1Rs in VgluT2-expressing glutamatergic neurons (VgluT2-CB1 -/-) and Cre-dependent viral vector to express light-sensitive channelrhodopsin-2 into VTA glutamatergic neurons. We found that photoactivation of VTA glutamatergic neurons produced robust intracranial self-stimulation (ICSS) behavior, which was dose-dependently blocked by DA receptor antagonists, but enhanced by cocaine. In contrast, Δ9-tetrahydrocannabinol (Δ9-THC), the major psychoactive component of cannabis, produced dose-dependent conditioned place aversion and a reduction in the above optical ICSS in VgluT2-cre control mice, but not in VgluT2-CB1 -/- mice. These findings suggest that activation of CB1Rs in VgluT2-expressing glutamate neurons produces aversive effects that might explain why cannabinoid is not rewarding in rodents and might also account for individual differences in the hedonic effects of cannabis in humans.
Cannabinoid CB2 receptors are expressed in glutamate neurons in the red nucleus and functionally modulate motor behavior in mice
Zhang, HY;Shen, H;Gao, M;Ma, Z;Hempel, B;Bi, GH;Gardner, EL;Wu, J;Xi, ZX;
PMID: 33789118 | DOI: 10.1016/j.neuropharm.2021.108538
Cannabinoids produce a number of central nervous system effects via the CB2 receptor (CB2R), including analgesia, antianxiety, anti-reward, hypoactivity and attenuation of opioid-induced respiratory depression. However, the cellular distributions of the CB2Rs in the brain remain unclear. We have reported that CB2Rs are expressed in midbrain dopamine (DA) neurons and functionally regulate DA-mediated behavior(s). Unexpectedly, high densities of CB2-like signaling were also found in a neighboring motor structure - the red nucleus (RN) of the midbrain. In the present study, we systematically explored CB2R expression and function in the RN. Immunohistochemistry and in situ hybridization assays showed high densities of CB2R-immunostaining and mRNA signal in RN magnocellular glutamate neurons in wildtype and CB1-knockout, but not CB2-knockout, mice. Ex vivo electrophysiological recordings in midbrain slices demonstrated that CB2R activation by JWH133 dose-dependently inhibited firing rates of RN magnocellular neurons in wildtype, but not CB2-knockout, mice, while having no effect on RN GABA neurons in transgenic GAD67-GFP reporter mice, suggesting CB2-mediated effects on glutamatergic neurons. In addition, microinjection of JWH133 into the RN produced robust ipsilateral rotations in wildtype, but not CB2-knockout mice, which was blocked by pretreatment with either a CB2 or DA D1 or D2 receptor antagonist, suggesting a DA-dependent effect. Finally, fluorescent tract tracing revealed glutamatergic projections from the RN to multiple brain areas including the ventral tegmental area, nucleus accumbens, thalamus, and cerebellum. These findings suggest that CB2Rs in RN glutamate neurons functionally modulate motor activity, and therefore, constitute a new target in cannabis-based medication development for motor disorders.
Zalachoras, I;Astori, S;Meijer, M;Grosse, J;Zanoletti, O;de Suduiraut, IG;Deussing, JM;Sandi, C;
PMID: 35319997 | DOI: 10.1126/sciadv.abj9019
Individuals frequently differ in their behavioral and cognitive responses to stress. However, whether motivation is differently affected by acute stress in different individuals remains to be established. By exploiting natural variation in trait anxiety in outbred Wistar rats, we show that acute stress facilitates effort-related motivation in low anxious animals, while dampening effort in high anxious ones. This model allowed us to address the mechanisms underlying acute stress-induced differences in motivated behavior. We show that CRHR1 expression levels in dopamine neurons of the ventral tegmental area (VTA)-a neuronal type implicated in the regulation of motivation-depend on animals' anxiety, and these differences in CRHR1 expression levels explain the divergent effects of stress on both effortful behavior and the functioning of mesolimbic DA neurons. These findings highlight CRHR1 in VTA DA neurons-whose levels vary with individuals' anxiety-as a switching mechanism determining whether acute stress facilitates or dampens motivation.
Porcu, A;Nilsson, A;Booreddy, S;Barnes, SA;Welsh, DK;Dulcis, D;
PMID: 36054362 | DOI: 10.1126/sciadv.abn9867
Seasonal changes in day length (photoperiod) affect numerous physiological functions. The suprachiasmatic nucleus (SCN)-paraventricular nucleus (PVN) axis plays a key role in processing photoperiod-related information. Seasonal variations in SCN and PVN neurotransmitter expression have been observed in humans and animal models. However, the molecular mechanisms by which the SCN-PVN network responds to altered photoperiod is unknown. Here, we show in mice that neuromedin S (NMS) and vasoactive intestinal polypeptide (VIP) neurons in the SCN display photoperiod-induced neurotransmitter plasticity. In vivo recording of calcium dynamics revealed that NMS neurons alter PVN network activity in response to winter-like photoperiod. Chronic manipulation of NMS neurons is sufficient to induce neurotransmitter switching in PVN neurons and affects locomotor activity. Our findings reveal previously unidentified molecular adaptations of the SCN-PVN network in response to seasonality and the role for NMS neurons in adjusting hypothalamic function to day length via a coordinated multisynaptic neurotransmitter switching affecting behavior.
