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.
Mou, TM;Lane, MV;Ireland, DDC;Verthelyi, D;Tonelli, LH;Clark, SM;
PMID: 35995342 | DOI: 10.1016/j.nbd.2022.105840
An early inflammatory insult is the most recognized risk factor associated with neurodevelopmental psychiatric disorders, even more so than genetic variants. Notably, complement component 4 (C4), a molecule involved in inflammatory responses, has been strongly associated with schizophrenia (SZ) and its role in other neurodevelopmental disorders, such as autism (ASD), is an area of active investigation. However, while C4 in SZ has been implicated in the context of synaptic pruning, little is known about its neuroinflammatory role. The subventricular zone (SVZ) is a region heavily involved in neurodevelopment and neuroimmune interactions through the lifespan; thus, it is a region wherein C4 may play a vital role in disease pathology. Using in situ hybridization with radioactive riboprobes and RNAscope, we identified robust astrocytic expression of C4 in the SVZ and in the septum pellucidum. C4 was also expressed in ependyma, neurons, and Ki67+ progenitor cells. Examination of mRNA levels showed elevated C4 in both ASD and SZ, with higher expression in SZ compared to controls. Targeted transcriptomic analysis of inflammatory pathways revealed a strong association of complement system genes with SZ, and to a lesser extent, ASD, as well as generalized immune dysregulation without a strong association with known infectious pathways. Analysis of differentially expressed genes (DEGs) showed that ASD DEGs were enriched in adaptive immune system functions such as Th cell differentiation, while SZ DEGs were enriched in innate immune system functions, including NF-κB and toll like receptor signaling. Moreover, the number of Ki67+ cells was significantly higher in ASD compared to SZ and controls. Taken together, these results support a role for C4 into inflammatory-neuroimmune dysregulation observed in SZ and ASD pathology.
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.
Underwood, CF;Burke, PGR;Kumar, NN;Goodchild, AK;McMullan, S;Phillips, JK;Hildreth, CM;
PMID: 35654013 | DOI: 10.1159/000525337
Angiotensin (Ang) II signalling in the hypothalamic paraventricular nucleus (PVN) via angiotensin type-1a receptors (AT1R) regulates vasopressin release and sympathetic nerve activity - two effectors of blood pressure regulation. We determined the cellular expression and function of AT1R in the PVN of a rodent model of polycystic kidney disease (PKD), the Lewis Polycystic Kidney (LPK) rat, to evaluate its contribution to blood pressure regulation and augmented vasopressin release in PKD.PVN AT1R gene expression was quantified with fluorescent in-situ hybridisation in LPK and control rats. PVN AT1R function was assessed with pharmacology under urethane anaesthesia in LPK and control rats instrumented to record arterial pressure and sympathetic nerve activity.AT1R gene expression was upregulated in the PVN, particularly in CRH neurons, of LPK versus control rats. PVN microinjection of Ang II produced larger increases in systolic blood pressure in LPK versus control rats (36±5 vs. 17±2 mmHg; P<0.01). Unexpectedly, Ang II produced regionally heterogeneous sympathoinhibition (renal: -33%; splanchnic: -12%; lumbar no change) in LPK and no change in controls. PVN pre-treatment with losartan, a competitive AT1R antagonist, blocked the Ang II-mediated renal sympathoinhibition and attenuated the pressor response observed in LPK rats. The Ang II pressor effect was also blocked by systemic OPC-21268, a competitive V1A receptor antagonist, but unaffected by hexamethonium, a sympathetic ganglionic blocker.Collectively, our data suggest that upregulated AT1R expression in PVN sensitises neuroendocrine release of vasopressin in the LPK, identifying a central mechanism for the elevated vasopressin levels present in PKD.The Author(s).
Yang, TS;Reichard, MV;Thomas, JE;Miller, LS;Marr, HS;
Cytauxzoon felis is a tick-borne piroplasmid hemoparasite that causes life-threatening disease in cats. Despite the critical role that ticks play in disease transmission and development, our knowledge regarding the C. felis life cycle remains limited to the feline hosts and no stage of the parasite has been identified or investigated in ticks. Sporozoites are the infectious stage of piroplasmids that are transmitted by ticks. In other tick-borne piroplasmids, sporozoites have played a key role in disease prevention and management. We believe sporozoites have similar potential for cytauxzoonosis. Therefore, the objective of this study is to evaluate different molecular and microscopic techniques to detect C. felis sporozoites in tick salivary glands (SG). A total of 140 Amblyomma americanum ticks that were fed on C. felis-infected cats as nymphs were included for this study. Specifically, dissected SGs were quartered and subjected to C. felis RT-PCR, RNAscope in situ hybridization (ISH), histology, direct azure staining, and transmission electron microscopy (TEM). Cytauxzoon felis RT-PCR was also performed on half tick (HT) carcasses after SG dissection. Cytauxzoon felis RNA was detected in SGs of 17 ticks. Of these, 7 ticks had microscopic visualization via ISH and/or TEM. The remaining 10 ticks had only molecular detection of C. felis in SGs via RT-PCR without visualization. Cytauxzoon felis RNA was detected solely in HT carcasses via RT-PCR in 9 additional ticks. In ISH-positive tick SGs, hybridization signals were present in cytoplasms of SG acinar cells. TEM captured rare C. felis organisms with characteristic ultrastructural features of piroplasmid parasites. This study describes the first direct visualization of any developing stage of C. felis in ticks. Forthcoming studies should employ a combination of molecular and microscopic techniques to investigate the C. felis life cycle in A. americanum.
Kashima DT, Grueter BA.
PMID: 28760987 | DOI: 10.1073/pnas.1705974114
Behavioral manifestations of drug-seeking behavior are causally linked to alterations of synaptic strength onto nucleus accumbens (NAc) medium spiny neurons (MSN). Although neuron-driven changes in physiology and behavior are well characterized, there is a lack of knowledge of the role of the immune system in mediating such effects. Toll-like receptor 4 (TLR4) is a pattern recognition molecule of the innate immune system, and evidence suggests that it modulates drug-related behavior. Using TLR4 knockout (TLR4.KO) mice, we show that TLR4 plays a role in NAc synaptic physiology and behavior. In addition to differences in the pharmacological profile of N-methyl-d-aspartate receptors (NMDAR) in the NAc core, TLR4.KO animals exhibit a deficit in low-frequency stimulation-induced NMDAR-dependent long-term depression (LTD). Interestingly, the synaptic difference is region specific as no differences were found in excitatory synaptic properties in the NAc shell. Consistent with altered NAc LTD, TLR4.KO animals exhibit an attenuation in drug reward learning. Finally, we show that TLR4 in the NAc core is primarily expressed on microglia. These results suggest that TLR4 influences NAc MSN synaptic physiology and drug reward learning and behavior.
Li S, Uno Y, Rudolph U, Cobb J, Liu J, Anderson T, Levy D, Balu DT, Coyle JT.
PMID: 29305854 | DOI: 10.1016/j.bcp.2017.12.023
D-Serine is a co-agonist at forebrain N-methyl-D-aspartate receptors (NMDAR) and is synthesized by serine racemase (SR). Although D-serine and SR were originally reported to be localized to glia, recent studies have provided compelling evidence that under healthy physiologic conditions both are localized primarily in neurons. However, in pathologic conditions, reactive astrocytes can also express SR and synthesize D-serine. Since cultured astrocytes exhibit features of reactive astrocytes, we have characterized D-serine synthesis and the expression of enzymes involved in its disposition in primary glial cultures. The levels of SR were quite low early in culture and increased markedly in all astrocytes with the duration in vitro. The concentration of D-serine in the culture medium increased in parallel with SR expression in the astrocytes. Microglia, identified by robust expression of Iba1, did not express SR. While the levels of glial fibrillary acidic protein (GFAP), glycine decarboxylase (GLDC) and phosphoglycerate dehydrogenase (PHGDH), the initial enzyme in the pathway converting glycine to L-serine, remained constant in culture, the expression of lipocalin-2, a marker for pan-reactive astrocytes, increased several-fold. The cultured astrocytes also expressed Complement-3a, a marker for a subpopulation of reactive astrocytes (A1). Astrocytes grown from mice with a copy number variant associated with psychosis, which have four copies of the GLDC gene, showed a more rapid production of D-serine and a reduction of glycine in the culture medium. These results substantiate the conclusion that A1 reactive astrocytes express SR and release D-serine under pathologic conditions, which may contribute to their neurotoxic effects by activating extra-synaptic NMDARs.
Key role for hypothalamic interleukin-6 in food-motivated behavior and body weight regulation
López-Ferreras, L;Longo, F;Richard, J;Eerola, K;Shevchouk, O;Tuzinovic, M;Skibicka, K;
| DOI: 10.1016/j.psyneuen.2021.105284
The pro-inflammatory role of interleukin-6 (IL-6) is well-characterized. Blockade of IL-6, by Tocilizumab, is used in patients with rheumatoid arthritis and those diagnosed with cytokine storm. However, brain-produced IL-6 has recently emerged as a critical mediator of gut/adipose communication with the brain. Central nervous system (CNS) IL-6 is engaged by peripheral and central signals regulating energy homeostasis. IL-6 is critical for mediating hypophagia and weight loss effects of a GLP-1 analog, exendin-4, a clinically utilized drug. However, neuroanatomical substrates and behavioral mechanisms of brain IL-6 energy balance control remain poorly understood. We propose that the lateral hypothalamus (LH) is an IL-6-harboring brain region, key to food intake and food reward control. Microinjections of IL-6 into the LH reduced chow and palatable food intake in male rats. In contrast, female rats responded with reduced motivated behavior for sucrose, measured by the progressive ratio operant conditioning test, a behavioral mechanism previously not linked to IL-6. To test whether IL-6, produced in the LH, is necessary for ingestive and motivated behaviors, and body weight homeostasis, virogenetic knockdown by infusion of AAV-siRNA-IL6 into the LH was utilized. Attenuation of LH IL-6 resulted in a potent increase in sucrose-motivated behavior, without any effect on ingestive behavior or body weight in female rats. In contrast, the treatment did not affect any parameters measured (chow intake, sucrose-motivated behavior, locomotion, and body weight) in chow-fed males. However, when challenged with a high-fat/high-sugar diet, the male LH IL-6 knockdown rats displayed rapid weight gain and hyperphagia. Together, our data suggest that LH-produced IL-6 is necessary and sufficient for ingestive behavior and weight homeostasis in male rats. In females, IL-6 in the LH plays a critical role in food-motivated, but not ingestive behavior control or weight regulation. Thus, collectively these data support the idea that brain-produced IL-6 engages the hypothalamus to control feeding behavior.
Shi MM, Fan KM, Qiao YN, Xu JH, Qiu LJ, Li X, Liu Y, Qian ZQ, Wei CL, Han J, Fan J, Tian YF, Ren W, Liu ZQ.
PMID: 31142818 | DOI: 10.1038/s41380-019-0435-z
Stressful life events induce abnormalities in emotional and cognitive behaviour. The endogenous opioid system plays an essential role in stress adaptation and coping strategies. In particular, the µ-opioid receptor (μR), one of the major opioid receptors, strongly influences memory processing in that alterations in μR signalling are associated with various neuropsychiatric disorders. However, it remains unclear whether μR signalling contributes to memory impairments induced by acute stress. Here, we utilized pharmacological methods and cell-type-selective/non-cell-type-selective μR depletion approaches combined with behavioural tests, biochemical analyses, and in vitro electrophysiological recordings to investigate the role of hippocampal μR signalling in memory-retrieval impairment induced by acute elevated platform (EP) stress in mice. Biochemical and molecular analyses revealed that hippocampal μRs were significantly activated during acute stress. Blockage of hippocampal μRs, non-selective deletion of μRs or selective deletion of μRs on GABAergic neurons (μRGABA) reversed EP-stress-induced impairment of memory retrieval, with no effect on the elevation of serum corticosterone after stress. Electrophysiological results demonstrated that stress depressed hippocampal GABAergic synaptic transmission to CA1 pyramidal neurons, thereby leading to excitation/inhibition (E/I) imbalance in a μRGABA-dependent manner. Pharmaceutically enhancing hippocampal GABAAreceptor-mediated inhibitory currents in stressed mice restored their memory retrieval, whereas inhibiting those currents in the unstressed mice mimicked the stress-induced impairment of memory retrieval. Our findings reveal a novel pathway in which endogenous opioids recruited by acute stress predominantly activate μRGABA to depress GABAergic inhibitory effects on CA1 pyramidal neurons, which subsequently alters the E/I balance in the hippocampus and results in impairment of memory retrieval.
Yosten GL, Harada CM, Haddock CJ, Giancotti LA, Kolar GR, Patel R, Guo C, Chen Z, Zhang J, Doyle TM, Dickenson AH, Samson WK, Salvemini D.
PMID: 31999650 | DOI: 10.1172/JCI133270
Treating neuropathic pain is challenging and novel non-opioid based medicines are needed. Using unbiased receptomics, transcriptomic analyses, immunofluorescence and in situ hybridization, we found the expression of the orphan GPCR (oGPCR) Gpr160 and GPR160 increased in the rodent dorsal horn of the spinal cord (DH-SC) following traumatic nerve injury. Genetic and immunopharmacological approaches demonstrated that GPR160 inhibition in the spinal cord prevented and reversed neuropathic pain in male and female rodents without altering normal pain response. GPR160 inhibition in the spinal cord attenuated sensory processing in the thalamus, a key relay in the sensory discriminative pathways of pain. We also identified cocaine- and amphetamine-regulated transcript peptide (CARTp) as a GPR160 ligand. Inhibiting endogenous CARTp signaling in spinal cord attenuated neuropathic pain, whereas exogenous intrathecal (i.th.) CARTp evoked painful hypersensitivity through GPR160-dependent ERK and cAMP response element-binding protein (CREB). Our findings de-orphanize GPR160, identify it as a determinant of neuropathic pain and potential therapeutic target, and provide insights to its signaling pathways. CARTp is involved in many diseases including depression, reward and addiction, de-orphanization of GPR160 is a major step forward understanding the role of CARTp signaling in health and disease
Griffiths PR, Lolait SJ, Bijabhai A, O'Carroll-Lolait A, Paton JFR, O'Carroll AM
PMID: 32315363 | DOI: 10.1371/journal.pone.0231844
The vascular organ of the lamina terminalis, subfornical organ (SFO), and area postrema comprise the sensory circumventricular organs (CVO) which are central structures that lie outside the blood brain barrier and are thought to provide an interface between peripherally circulating signals and the brain through their projections to central autonomic structures. The SFO expresses mRNA for the G protein-coupled apelin receptor (APJ, gene name aplnr) and exogenous microinjection of the neuropeptide apelin (apln) to the SFO elicits a depressor effect. Here we investigated the expression and cellular distribution of aplnr, apln and the recently described ligand apela (apela) in the CVOs and investigated whether differences in the levels of expression of apelinergic gene transcripts in these regions might underlie the chronic elevated blood pressure seen in hypertension. We carried out multiplex in situ hybridization histochemistry on CVO tissue sections from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) controls. Confocal immunofluorescent images indicated strong aplnr expression, with lower levels of apln and modest apela expression, in the CVOs of both WKY rats and SHRs, in both neurons and glia. The expression level of aplnr transcripts was increased in the SFO of SHRs compared to WKY rats. Our data may highlight a potential dysfunction in the communication between CVOs and downstream signalling pathways in SHRs, which may contribute to its different phenotype/s
Futrega, K;Shajib, M;Robey, P;Doran, M;
| DOI: 10.3390/organoids2020008
(1) Background: There are no high-throughput microtissue platforms for generating bone marrow micro-ossicles. Herein, we describe a method for the assembly of arrays of microtissues from bone marrow stromal cells (BMSC) in vitro and their maturation into bone marrow micro-ossicles in vivo. (2) Methods: Discs with arrays of 50 microwells were used to assemble microtissues from 3 × 105 BMSCs each on a nylon mesh carrier. Microtissues were cultured in chondrogenic induction medium followed by hypertrophic medium in an attempt to drive endochondral ossification, and then they were implanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, where they were remodeled into bone marrow micro-ossicles. Mice were transplanted with 105 human umbilical cord blood CD34+ cells. (3) Results: Micro-ossicles contained more human CD45+ cells, but fewer human CD34+ progenitor cells than mouse marrow. Human hematopoietic progenitor cells cycle rapidly at non-physiological rates in mouse marrow, and reduced CD34+ cell content in micro-ossicles is consistent with the notion that the humanized niche better controls progenitor cell cycling. (4) Conclusions: Assembling microtissues in microwells, linked by a nylon membrane carrier, provides an elegant method to manufacture and handle arrays of microtissues with bone organ-like properties. More generally, this approach and platform could aid bridging the gap between in vitro microtissue manipulation and in vivo microtissue implantation.
