Himmel LE, Hackett TA, Moore JL, Adams WR, Thomas G, Novitskaya T, Caprioli RM, Zijlstra A, Mahadevan-Jansen A, Boyd KL.
PMID: 30462242 | DOI: 10.1093/ilar/ily004
For decades, histopathology with routine hematoxylin and eosin staining has been and remains the gold standard for reaching a morphologic diagnosis in tissue samples from humans and veterinary species. However, within the past decade, there has been exponential growth in advanced techniques for in situ tissue biomarker imaging that bridge the divide between anatomic and molecular pathology. It is now possible to simultaneously observe localization and expression magnitude of multiple protein, nucleic acid, and molecular targets in tissue sections and apply machine learning to synthesize vast, image-derived datasets. As these technologies become more sophisticated and widely available, a team-science approach involving subspecialists with medical, engineering, and physics backgrounds is critical to upholding quality and validity in studies generating these data. The purpose of this manuscript is to detail the scientific premise, tools and training, quality control, and data collection and analysis considerations needed for the most prominent advanced imaging technologies currently applied in tissue sections: immunofluorescence, in situ hybridization, laser capture microdissection, matrix-assisted laser desorption ionization imaging mass spectrometry, and spectroscopic/optical methods. We conclude with a brief overview of future directions for ex vivo and in vivo imaging techniques.
Noh, YW;Yook, C;Kang, J;Lee, S;Kim, Y;Yang, E;Kim, H;Kim, E;
PMID: 35982261 | DOI: 10.1038/s42003-022-03813-y
IRSp53 (or BAIAP2) is an abundant excitatory postsynaptic scaffolding/adaptor protein that is involved in actin regulation and has been implicated in autism spectrum disorders, schizophrenia, and attention-deficit/hyperactivity disorder. IRSp53 deletion in mice leads to enhanced NMDA receptor (NMDAR) function and social deficits that are responsive to NMDAR inhibition. However, it remains unclear whether IRSp53 re-expression in the adult IRSp53-mutant mouse brain after the completion of brain development could reverse these synaptic and behavioral dysfunctions. Here we employed a brain-blood barrier (BBB)-penetrant adeno-associated virus (AAV) known as PHP.eB to drive adult IRSp53 re-expression in IRSp53-mutant mice. The adult IRSp53 re-expression normalized social deficits without affecting hyperactivity or anxiety-like behavior. In addition, adult IRSp53 re-expression normalized NMDAR-mediated excitatory synaptic transmission in the medial prefrontal cortex. Our results suggest that adult IRSp53 re-expression can normalize synaptic and behavioral deficits in IRSp53-mutant mice and that BBB-penetrant adult gene re-expression has therapeutic potential.
Molecular therapy : the journal of the American Society of Gene Therapy
Tadokoro, T;Bravo-Hernandez, M;Agashkov, K;Kobayashi, Y;Platoshyn, O;Navarro, M;Marsala, S;Miyanohara, A;Yoshizumi, T;Shigyo, M;Krotov, V;Juhas, S;Juhasova, J;Nguyen, D;Kupcova Skalnikova, H;Motlik, J;Studenovska, H;Proks, V;Reddy, R;Driscoll, SP;Glenn, TD;Kemthong, T;Malaivijitnond, S;Tomori, Z;Vanicky, I;Kakinohana, M;Pfaff, SL;Ciacci, J;Belan, P;Marsala, M;
PMID: 35524407 | DOI: 10.1016/j.ymthe.2022.04.023
Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.
Sartori, AM;Hofer, AS;Scheuber, MI;Rust, R;Kessler, TM;Schwab, ME;
PMID: 34826427 | DOI: 10.1016/j.expneurol.2021.113937
Neurogenic lower urinary tract dysfunction typically develops after spinal cord injury. We investigated the time course and the anatomical changes in the spinal cord that may be causing lower urinary tract symptoms following injury. Rats were implanted with a bladder catheter and external urethral sphincter electromyography electrodes. Animals underwent a large, incomplete spinal transection at the T8/9 spinal level. At 1, 2-3, and 4 weeks after injury, the animals underwent urodynamic investigations. Urodynamic investigations showed detrusor overactivity and detrusor-sphincter-dyssynergia appearing over time at 3-4 weeks after injury. Lower urinary tract dysfunction was accompanied by an increase in density of C-fiber afferents in the lumbosacral dorsal horn. CRF-positive Barrington's and 5-HT-positive bulbospinal projections drastically decreased after injury, with partial compensation for the CRF fibers at 3-4 weeks. Interestingly, a decrease over time was observed in the number of GABAergic neurons in the lumbosacral dorsal horn and lamina X, and a decrease of glutamatergic cells in the dorsal horn. Detrusor overactivity and detrusor-sphincter-dyssynergia might therefore arise from a discrepancy in inhibitory/excitatory interneuron activity in the lumbosacral cord as well as input changes which develop over time after injury. The processes point to spinal plastic changes leading to malfunction of the important physiological pathway of lower urinary tract control.
Leithead, AB;Godino, A;Barbier, M;Harony-Nicolas, H;
PMID: 37245781 | DOI: 10.1016/j.biopsych.2023.05.016
The posterior intralaminar (PIL) complex of the thalamus is a multimodal nucleus that has been implicated in maternal behaviors and conspecific social behaviors in male and female rodents. Glutamatergic neurons are a major component of the PIL; however, their specific activity and role during social interactions has not yet been assessed.We used immunohistochemistry for the immediate early gene c-fos as a proxy for neuronal activity in the PIL of mice exposed to a novel social stimulus, a novel object stimulus, or no stimulus. We then used fiber photometry to record neural activity of glutamatergic neurons in the PIL in real-time during social and non-social interactions. Finally, we used inhibitory DREADDs in glutamatergic PIL neurons and tested social preference and social habituation-dishabituation.We observed significantly more c-fos-positive cells in the PIL of mice exposed to social versus object or no stimuli. Neural activity of PIL glutamatergic neurons was increased when male and female mice were engaged in social interaction with a same-sex juvenile or opposite-sex adult, but not a toy mouse. Neural activity positively correlated with social investigation bout length and negatively correlated with chronological order of bouts. Social preference was unaffected by inhibition; however, inhibiting activity of glutamatergic neurons in the PIL delayed the time it took female mice to form social habituation.Together these findings suggest that glutamatergic PIL neurons respond to social stimuli in both male and female mice and may regulate perceptual encoding of social information to facilitate recognition of social stimuli.
Proc Natl Acad Sci U S A.
Meng D, Li HQ, Deisseroth K, Leutgeb S, Spitzer NC.
PMID: 29686073 | DOI: 10.1073/pnas.1801598115
Neurotransmitter switching in the adult mammalian brain occurs following photoperiod-induced stress, but the mechanism of regulation is unknown. Here, we demonstrate that elevated activity of dopaminergic neurons in the paraventricular nucleus of the hypothalamus (PaVN) in the adult rat is required for the loss of dopamine expression after long-day photoperiod exposure. The transmitter switch occurs exclusively in PaVN dopaminergic neurons that coexpress vesicular glutamate transporter 2 (VGLUT2), is accompanied by a loss of dopamine type 2 receptors (D2Rs) on corticotrophin-releasing factor (CRF) neurons, and can lead to increased release of CRF. Suppressing activity of all PaVN glutamatergic neurons decreases the number of inhibitory PaVN dopaminergic neurons, indicating homeostatic regulation of transmitter expression in the PaVN.
The Journal of neuroscience : the official journal of the Society for Neuroscience
Fudge, JL;Kelly, EA;Hackett, TA;
PMID: 36280261 | DOI: 10.1523/JNEUROSCI.1453-22.2022
The central extended amygdala (CEA) and ventral pallidum (VP) are involved in diverse motivated behaviors based on rodent models. These structures are conserved, but expanded, in higher primates including human. Corticotropin releasing factor (CRF), a canonical 'stress molecule' associated with the CEA and VP circuitry across species, is dynamically regulated by stress and drugs of abuse and misuse. CRF's effects on circuits critically depend on its colocation with primary 'fast' transmitters, making this crucial for understanding circuit effects. We surveyed the distribution and colocalization of CRF-, VGluT2- (vesicular glutamate transporter 2) and VGAT- (vesicular GABA transporter) mRNA in specific subregions of the CEA and VP in young male monkeys. Although CRF-containing neurons were clustered in the lateral central bed nucleus (BSTLcn), the majority were broadly dispersed throughout other CEA subregions, and the VP. CRF/VGAT-only neurons were highest in the BSTLcn, lateral central amygdala nucleus (CeLcn), and medial central amygdala nucleus (CeM) (74%, 73%, and 85%, respectively). In contrast, lower percentages of CRF/VGAT only neurons populated the sublenticular extended amygdala (SLEAc), ventrolateral bed nucleus (BSTLP), and VP (53%, 54%, 17%, respectively), which had higher complements of CRF/VGAT/VGluT2 labeled neurons (33%, 29%, 67%, respectively). Thus, the majority of CRF-neurons at the 'poles' (BSTLcn and CeLcn/CeM) of the CEA are inhibitory, while the 'extended' BSTLP and SLEAc subregions, and neighboring VP, have a more complex profile with admixtures of 'multiplexed' excitatory CRF neurons. CRF's colocalization with its various fast transmitters is likely circuit-specific, and relevant for understanding CRF actions on specific target sites.SIGNIFICANCE STATEMENT:The central extended amygdala (CEA) and ventral pallidum (VP) regulate multiple motivated behaviors through differential downstream projections. The stress neuropeptide corticotropin releasing factor (CRF) is enriched in the CEA, and is thought to 'set the gain' through modulatory effects on co-expressed primary transmitters. Using protein and transcript assays in monkey, we found that CRF neurons are broadly and diffusely distributed in CEA and VP. CRF mRNA+ neurons colocalize with VGAT (GABA) and VGluT2 (glutamate) mRNAs in different proportions depending on subregion. CRF mRNA was also co-expressed in a subpopulation of VGAT/VGluT2 mRNA ('multiplexed') cells which were most prominent in the VP and 'pallidal'-like parts of the CEA. Heterogeneous CRF and fast transmitter co-expression across CEA/VP subregions implies circuit-specific effects.
Proc Natl Acad Sci U S A.
Shen H, Marino RAM, McDevitt RA, Bi GH, Chen K, Madeo G, Lee PT, Liang Y, De Biase LM, Su TP, Xi ZX, Bonci A.
PMID: 30442663 | DOI: 10.1073/pnas.1800886115
A subset of midbrain dopamine (DA) neurons express vesicular glutamate transporter 2 (VgluT2), which facilitates synaptic vesicle loading of glutamate. Recent studies indicate that such expression can modulate DA-dependent reward behaviors, but little is known about functional consequences of DA neuron VgluT2 expression in neurodegenerative diseases like Parkinson's disease (PD). Here, we report that selective deletion of VgluT2 in DA neurons in conditional VgluT2-KO (VgluT2-cKO) mice abolished glutamate release from DA neurons, reduced their expression of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB), and exacerbated the pathological effects of exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, viral rescue of VgluT2 expression in DA neurons of VglutT2-cKO mice restored BDNF/TrkB expression and attenuated MPTP-induced DA neuron loss and locomotor impairment. Together, these findings indicate that VgluT2 expression in DA neurons is neuroprotective. Genetic or environmental factors causing reduced expression or function of VgluT2 in DA neurons may place some individuals at increased risk for DA neuron degeneration. Therefore, maintaining physiological expression and function of VgluT2 in DA neurons may represent a valid molecular target for the development of preventive therapeutic interventions for PD.
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.
Yan, JJ;Ding, XJ;He, T;Chen, AX;Zhang, W;Yu, ZX;Cheng, XY;Wei, CY;Hu, QD;Liu, XY;Zhang, YL;He, M;Xie, ZY;Zha, X;Xu, C;Cao, P;Li, H;Xu, XH;
PMID: 36463200 | DOI: 10.1038/s41467-022-35211-7
Behavioral observations suggest a connection between anxiety and predator defense, but the underlying neural mechanisms remain unclear. Here we examine the role of the anterior hypothalamic nucleus (AHN), a node in the predator defense network, in anxiety-like behaviors. By in vivo recordings in male mice, we find that activity of AHN GABAergic (AHNVgat+) neurons shows individually stable increases when animals approach unfamiliar objects in an open field (OF) or when they explore the open-arm of an elevated plus-maze (EPM). Moreover, object-evoked AHN activity overlap with predator cue responses and correlate with the object and open-arm avoidance. Crucially, exploration-triggered optogenetic inhibition of AHNVgat+ neurons reduces object and open-arm avoidance. Furthermore, retrograde viral tracing identifies the ventral subiculum (vSub) of the hippocampal formation as a significant input to AHNVgat+ neurons in driving avoidance behaviors in anxiogenic situations. Thus, convergent activation of AHNVgat+ neurons serves as a shared mechanism between anxiety and predator defense to promote behavioral avoidance.
Ilanges, A;Shiao, R;Shaked, J;Luo, JD;Yu, X;Friedman, JM;
PMID: 36071158 | DOI: 10.1038/s41586-022-05161-7
Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.
Lewis, EM;Spence, HE;Akella, N;Buonanno, A;
PMID: 36075962 | DOI: 10.1038/s41380-022-01747-9
Prefrontal cortex (PFC) is a site of information convergence important for behaviors relevant to psychiatric disorders. Despite the importance of inhibitory GABAergic parvalbumin-expressing (PV+) interneurons to PFC circuit function and decades of interest in N-methyl-D-aspartate receptors (NMDARs) in these neurons, examples of defined circuit functions that depend on PV+ interneuron NMDARs have been elusive. Indeed, it remains controversial whether all PV+ interneurons contain functional NMDARs in adult PFC, which has major consequences for hypotheses of the pathogenesis of psychiatric disorders. Using a combination of fluorescent in situ hybridization, pathway-specific optogenetics, cell-type-specific gene ablation, and electrophysiological recordings from PV+ interneurons, here we resolve this controversy. We found that nearly 100% of PV+ interneurons in adult medial PFC (mPFC) express transcripts encoding GluN1 and GluN2B, and they have functional NMDARs. By optogenetically stimulating corticocortical and thalamocortical inputs to mPFC, we show that synaptic NMDAR contribution to PV+ interneuron EPSCs is pathway-specific, which likely explains earlier reports of PV+ interneurons without synaptic NMDAR currents. Lastly, we report a major contribution of NMDARs in PV+ interneurons to thalamus-mediated feedforward inhibition in adult mPFC circuits, suggesting molecular and circuit-based mechanisms for cognitive impairment under conditions of reduced NMDAR function. These findings represent an important conceptual advance that has major implications for hypotheses of the pathogenesis of psychiatric disorders.
