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.
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.
Lotun, A;Li, D;Xu, H;Su, Q;Tuncer, S;Sanmiguel, J;Mooney, M;Baer, CE;Ulbrich, R;Eyles, SJ;Strittmatter, L;Hayward, LJ;Gessler, DJ;Gao, G;
PMID: 37149081 | DOI: 10.1016/j.pneurobio.2023.102460
Myelinating oligodendrocytes are essential for neuronal communication and homeostasis of the central nervous system (CNS). One of the most abundant molecules in the mammalian CNS is N-acetylaspartate (NAA), which is catabolized into L-aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. The resulting acetate moiety is thought to contribute to myelin lipid synthesis. In addition, affected NAA metabolism has been implicated in several neurological disorders, including leukodystrophies and demyelinating diseases such as multiple sclerosis. Genetic disruption of ASPA function causes Canavan disease, which is hallmarked by increased NAA levels, myelin and neuronal loss, large vacuole formation in the CNS, and early death in childhood. Although NAA's direct role in the CNS is inconclusive, in peripheral adipose tissue, NAA-derived acetate has been found to modify histones, a mechanism known to be involved in epigenetic regulation of cell differentiation. We hypothesize that a lack of cellular differentiation in the brain contributes to the disruption of myelination and neurodegeneration in diseases with altered NAA metabolism, such as Canavan disease. Our study demonstrates that loss of functional Aspa in mice disrupts myelination and shifts the transcriptional expression of neuronal and oligodendrocyte markers towards less differentiated stages in a spatiotemporal manner. Upon re-expression of ASPA, these oligodendrocyte and neuronal lineage markers are either improved or normalized, suggesting that NAA breakdown by Aspa plays an essential role in the maturation of neurons and oligodendrocytes. Also, this effect of ASPA re-expression is blunted in old mice, potentially due to limited ability of neuronal, rather than oligodendrocyte, recovery.
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.
The Journal of experimental medicine
Hanuscheck, N;Thalman, C;Domingues, M;Schmaul, S;Muthuraman, M;Hetsch, F;Ecker, M;Endle, H;Oshaghi, M;Martino, G;Kuhlmann, T;Bozek, K;van Beers, T;Bittner, S;von Engelhardt, J;Vogt, J;Vogelaar, CF;Zipp, F;
PMID: 35587822 | DOI: 10.1084/jem.20211887
Evidence is emerging that immune responses not only play a part in the central nervous system (CNS) in diseases but may also be relevant for healthy conditions. We discovered a major role for the interleukin-4 (IL-4)/IL-4 receptor alpha (IL-4Rα) signaling pathway in synaptic processes, as indicated by transcriptome analysis in IL-4Rα-deficient mice and human neurons with/without IL-4 treatment. Moreover, IL-4Rα is expressed presynaptically, and locally available IL-4 regulates synaptic transmission. We found reduced synaptic vesicle pools, altered postsynaptic currents, and a higher excitatory drive in cortical networks of IL-4Rα-deficient neurons. Acute effects of IL-4 treatment on postsynaptic currents in wild-type neurons were mediated via PKCγ signaling release and led to increased inhibitory activity supporting the findings in IL-4Rα-deficient neurons. In fact, the deficiency of IL-4Rα resulted in increased network activity in vivo, accompanied by altered exploration and anxiety-related learning behavior; general learning and memory was unchanged. In conclusion, neuronal IL-4Rα and its presynaptic prevalence appear relevant for maintaining homeostasis of CNS synaptic function.
Samineni VK, Grajales-Reyes JG, Copits BA, O’Brien DE, Trigg SL, Gomez AM, Bruchas MR, Gereau RW.
PMID: - | DOI: 10.1523/ENEURO.0129-16.2017
The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pro-nociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here we demonstrate the different contributions of genetically-defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception.
Significance Statement The PAG is a midbrain region critical for the modulation of pain. However, the roles played by the distinct cell types within the PAG in nociceptive processing are poorly understood. This work addresses the divergent roles of glutamatergic and GABAergic PAG neuronal subpopulations in nociceptive processing. We demonstrate that activation of glutamatergic neurons or inhibition of GABAergic neurons suppresses nociception. Whereas inhibition of glutamatergic neuronal activity or activation of GABAergic neuronal activity potentiates nociception. This report identifies distinct roles for these neuronal populations in modulating nociceptive processing.
Cannabidiol produces distinct U-shaped dose-response effects on cocaine conditioned place preference and associated recruitment of prelimbic neurons in male rats
Biological Psychiatry Global Open Science
Nedelescu, H;Wagner, G;De Ness, G;Carrol, A;Kerr, T;Wang, J;Zhang, S;Chang, S;Than, A;Emerson, N;Suto, N;Weiss, F;
| DOI: 10.1016/j.bpsgos.2021.06.014
Background Cannabidiol (CBD) has received attention for the treatment of Substance Use Disorders. In preclinical models of relapse, CBD attenuates drug seeking across several drugs of abuse, including cocaine. However, in these models, CBD has not been consistently effective. This inconsistency in CBD effects may be related to presently insufficient information on the full spectrum of CBD dose effects on drug-related behaviors. Methods We address this issue by establishing a full dose-response profile of CBD’s actions using expression of cocaine-induced conditioned place preference (CPP) as a model for drug motivated behavior in male rats, and by concurrently identifying dose-dependent effects of CBD on underlying neuronal activation as well as distinct neuronal phenotypes showing dose-dependent activation changes. Additionally, CBD levels in plasma and brain were established. Results CBD produced linear increases in CBD brain/plasma concentrations but suppressed CPP in a distinct U-shaped manner. In parallel with its behavioral effects, CBD produced U-shaped suppressant effects on neuronal activation in the prelimbic but not infralimbic cortex or nucleus accumbens core and shell. RNAscope in situ hybridization identified suppression of glutamatergic and GABAergic signaling in the prelimbic cortex as a possible cellular mechanism for the attenuation of cocaine CPP by CBD. Conclusions The findings extend previous evidence on the potential of CBD in preventing drug motivated behavior. However, CBD’s dose-response profile may have important dosing implications for future clinical applications and may contribute to the understanding of discrepant CBD effects on drug seeking in the literature.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Ambler, M;Hitrec, T;Wilson, A;Cerri, M;Pickering, A;
PMID: 35440490 | DOI: 10.1523/JNEUROSCI.2102-21.2022
Torpor is a naturally occurring, hypometabolic, hypothermic state engaged by a wide range of animals in response to imbalance between the supply and demand for nutrients. Recent work has identified some of the key neuronal populations involved in daily torpor induction in mice, in particular projections from the preoptic area of the hypothalamus (POA) to the dorsomedial hypothalamus (DMH). The DMH plays a role in thermoregulation, control of energy expenditure, and circadian rhythms, making it well positioned to contribute to the expression of torpor. We used activity dependent genetic TRAPing techniques to target DMH neurons that were active during natural torpor bouts in female mice. Chemogenetic reactivation of torpor-TRAPed DMH neurons in calorie-restricted mice promoted torpor, resulting in longer and deeper torpor bouts. Chemogenetic inhibition of torpor-TRAPed DMH neurons did not block torpor entry, suggesting a modulatory role for the DMH in the control of torpor. This work adds to the evidence that the POA and the DMH form part of a circuit within the mouse hypothalamus that controls entry into daily torpor.SIGNIFICANCEDaily heterotherms such as mice employ torpor to cope with environments in which the supply of metabolic fuel is not sufficient for the maintenance of normothermia. Daily torpor involves reductions in body temperature, as well as active suppression of heart rate and metabolism. How the central nervous system controls this profound deviation from normal homeostasis is not known, but a projection from the preoptic area to the dorsomedial hypothalamus has recently been implicated. We demonstrate that the dorsomedial hypothalamus contains neurons that are active during torpor. Activity in these neurons promotes torpor entry and maintenance, but their activation alone does not appear to be sufficient for torpor entry.
Nikitin, P;Musina, G;Pekov, S;Kuzin, A;Popov, I;Belyaev, A;Kobyakov, G;Usachev, D;Nikolaev, V;Mikhailov, V;
| DOI: 10.3390/cancers15010145
Diffuse gliomas continue to be an important problem in neuro-oncology. To solve it, studies have considered the issues of molecular pathogenesis from the intratumoral heterogeneity point. Here, we carried out a comparative dynamic analysis of the different cell populations’ content in diffuse gliomas of different molecular profiles and grades, considering the cell populations’ functional properties and the relationship with patient survival, using flow cytometry, immunofluorescence, multiparametric fluorescent in situ hybridization, polymerase chain reaction, and cultural methods. It was shown that an increase in the IDH-mutant astrocytomas and oligodendrogliomas malignancy is accompanied by an increase in stem cells’ proportion and mesenchymal cell populations’ appearance arising from oligodendrocyte-progenitor-like cells with cell plasticity and cells’ hypoxia response programs’ activation. In glioblastomas, malignancy increase is accompanied by an increase in both stem and definitive cells with mesenchymal differentiation, while proneuronal glioma stem cells are the most likely the source of mesenchymal glioma stem cells, which, in hypoxic conditions, further give rise to mesenchymal-like cells. Clinical confirmation was a mesenchymal-like cell and mesenchymal glioma stem cell number, and the hypoxic and plastic molecular programs’ activation degree had a significant effect on relapse-free and overall survival. In general, we built a multi-vector model of diffuse gliomas’ pathogenetic tracing up to the practical plane.
Yu, B;Zhang, Q;Lin, L;Zhou, X;Ma, W;Wen, S;Li, C;Wang, W;Wu, Q;Wang, X;Li, XM;
PMID: 36788214 | DOI: 10.1038/s41421-022-00506-y
The amygdala, or an amygdala-like structure, is found in the brains of all vertebrates and plays a critical role in survival and reproduction. However, the cellular architecture of the amygdala and how it has evolved remain elusive. Here, we generated single-nucleus RNA-sequencing data for more than 200,000 cells in the amygdala of humans, macaques, mice, and chickens. Abundant neuronal cell types from different amygdala subnuclei were identified in all datasets. Cross-species analysis revealed that inhibitory neurons and inhibitory neuron-enriched subnuclei of the amygdala were well-conserved in cellular composition and marker gene expression, whereas excitatory neuron-enriched subnuclei were relatively divergent. Furthermore, LAMP5+ interneurons were much more abundant in primates, while DRD2+ inhibitory neurons and LAMP5+SATB2+ excitatory neurons were dominant in the human central amygdalar nucleus (CEA) and basolateral amygdalar complex (BLA), respectively. We also identified CEA-like neurons and their species-specific distribution patterns in chickens. This study highlights the extreme cell-type diversity in the amygdala and reveals the conservation and divergence of cell types and gene expression patterns across species that may contribute to species-specific adaptations.
Kiguchi, N;Ding, H;Park, SH;Mabry, KM;Kishioka, S;Shiozawa, Y;Alfonso Romero-Sandoval, E;Peters, CM;Ko, MC;
PMID: 35189108 | DOI: 10.1016/j.bcp.2022.114972
Despite accumulating evidence in rodents, the functional role of neuromedin B (NMB) in regulating somatosensory systems in primate spinal cord is unknown. We aimed to compare the expression patterns of NMB and its receptor (NMBR) and the behavioral effects of intrathecal (i.t.) NMB with gastrin-releasing peptide (GRP) on itch or pain in non-human primates (NHPs). We used six adult rhesus monkeys. The mRNA or protein expressions of NMB, GRP, and their receptors were evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, or in situ hybridization. We determined the behavioral effects of NMB or GRP via acute thermal nociception, capsaicin-induced thermal allodynia, and itch scratching response assays. NMB expression levels were greater than those of GRP in the dorsal root ganglia and spinal dorsal horn. Conversely, NMBR expression was significantly lower than GRP receptor (GRPR). I.t. NMB elicited only mild scratching responses, whereas GRP caused robust scratching responses. GRP- and NMB-elicited scratching responses were attenuated by GRPR (RC-3095) and NMBR (PD168368) antagonists, respectively. Moreover, i.t. NMB and GRP did not induce thermal hypersensitivity and GRPR and NMBR antagonists did not affect peripherally elicited thermal allodynia. Consistently, NMBR expression was low in both itch- and pain-responsive neurons in the spinal dorsal horn. Spinal NMB-NMBR system plays a minimal functional role in the neurotransmission of itch and pain in primates. Unlike the functional significance of the GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch.
