Septal GABA and Glutamate Neurons Express RXFP3 mRNA and Depletion of Septal RXFP3 Impaired Spatial Search Strategy and Long-Term Reference Memory in Adult Mice

Front Neuroanat

2019 Mar 08

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.

Neurocan regulates vulnerability to stress and the anti-depressant effect of ketamine in adolescent rats

Molecular psychiatry

2022 Mar 09

Yu, Z;Han, Y;Hu, D;Chen, N;Zhang, Z;Chen, W;Xue, Y;Meng, S;Lu, L;Zhang, W;Shi, J;
PMID: 35264728 | DOI: 10.1038/s41380-022-01495-w

Depression is more prevalent among adolescents than adults, but the underlying mechanisms remain largely unknown. Using a subthreshold chronic stress model, here we show that developmentally regulated expressions of the perineuronal nets (PNNs), and one of the components, Neurocan in the prelimbic cortex (PrL) are important for the vulnerability to stress and depressive-like behaviors in both adolescent and adult rats. Reduction of PNNs or Neurocan with pharmacological or viral methods to mimic the expression of PNNs in the PrL during adolescence compromised resilience to stress in adult rats, while virally mediated overexpression of Neurocan reversed vulnerability to stress in adolescent rats. Ketamine, a recent-approved drug for treatment-resistant depression rescued impaired function of Parvalbumin-positive neurons function, increased expression of PNNs in the PrL, and reversed depressive-like behaviors in adolescent rats. Furthermore, we show that Neurocan mediates the anti-depressant effect of ketamine, virally mediated reduction of Neurocan in the PrL abolished the anti-depressant effect of ketamine in adolescent rats. Our findings show an important role of Neurocan in depression in adolescence, and suggest a novel mechanism for the anti-depressant effect of ketamine.

Cell Type-Specific Gene Expression of Alpha 5 Subunit-Containing Gamma-Aminobutyric Acid Subtype A Receptors in Human and Mouse Frontal Cortex.

Molecular Neuropsychiatry

2019 Jan 23

Hu X,. Rocco BR, Fee C, Sibille E.
PMID: - | DOI: 10.1159/000495840

Converging evidence suggests that deficits in somatostatin (SST)-expressing neuron signaling contributes to major depressive disorder. Preclinical studies show that enhancing this signaling, specifically at α5 subunit-containing γ-aminobutyric acid subtype A receptors (α5-GABAARs), provides a potential means to overcome low SST neuron function. The cortical microcircuit comprises multiple subtypes of inhibitory γ-aminobutyric acid (GABA) neurons and excitatory pyramidal cells (PYCs). In this study, multilabel fluorescence in situ hybridization was used to characterize α5-GABAAR gene expression in PYCs and three GABAergic neuron subgroups – vasoactive intestinal peptide (VIP)-, SST-, and parvalbumin (PV)-expressing cells – in the human and mouse frontal cortex. Across species, we found the majority of gene expression in PYCs (human: 39.7%; mouse: 54.14%), less abundant expression in PV neurons (human: 20%; mouse: 16.33%), and no expression in VIP neurons (0%). Only human SST cells expressed GABRA5, albeit at low levels (human: 8.3%; mouse: 0%). Together, this localization suggests potential roles for α5-GABAARs within the cortical microcircuit: (1) regulators of PYCs, (2) regulators of PV cell activity across species, and (3) sparse regulators of SST cell inhibition in humans. These results will advance our ability to predict the effects of pharmacological agents targeting α5-GABAARs, which have shown therapeutic potential in preclinical animal models.

Context-Induced Reinstatement of Methamphetamine Seeking Is Associated with Unique Molecular Alterations in Fos-Expressing Dorsolateral Striatum Neurons

The Journal of Neuroscience, 8 April 2015, 35(14): 5625-5639

Rubio FJ, Liu QR, Li X, Cruz FC, Leão RM, Warren BL, Kambhampati S, Babin KR, McPherson KB, Cimbro R, Bossert JM, Shaham Y, Hope BT.
PMID: 25855177 | DOI: 10.1523/JNEUROSCI.4997-14.2015

Context-induced reinstatement of drug seeking is a well established animal model for assessing the neural mechanisms underlying context-induced drug relapse, a major factor in human drug addiction. Neural activity in striatum has previously been shown to contribute to context-induced reinstatement of heroin, cocaine, and alcohol seeking, but not yet for methamphetamine seeking. In this study, we found that context-induced reinstatement of methamphetamine seeking increased expression of the neural activity marker Fos in dorsal but not ventral striatum. Reversible inactivation of neural activity in dorsolateral but not dorsomedial striatum using the GABA agonists muscimol and baclofen decreased context-induced reinstatement. Based on our previous findings that Fos-expressing neurons play a critical role in conditioned drug effects, we assessed whether context-induced reinstatement was associated with molecular alterations selectively induced within context-activated Fos-expressing neurons. We used fluorescence-activated cell sorting to isolate reinstatement-activated Fos-positive neurons from Fos-negative neurons in dorsal striatum and used quantitative PCR to assess gene expression within these two populations of neurons. Context-induced reinstatement was associated with increased expression of the immediate early genes Fos and FosB and the NMDA receptor subunit gene Grin2a in only Fos-positive neurons. RNAscope in situ hybridization confirmed that Grin2a, as well as Grin2b, expression were increased in only Fos-positive neurons from dorsolateral, but not dorsomedial, striatum. Our results demonstrate an important role of dorsolateral striatum in context-induced reinstatement of methamphetamine seeking and that this reinstatement is associated with unique gene alterations in Fos-expressing neurons.

Maturation of GABAergic Synaptic Transmission From Neocortical Parvalbumin Interneurons Involves N-methyl-D-aspartate Receptor Recruitment of Cav2.1 Channels

Neuroscience

2023 Jan 20

Singh, M;Sapkota, K;Sakimura, K;Kano, M;Cowell, RM;Overstreet-Wadiche, L;Hablitz, JJ;Nakazawa, K;
PMID: 36682446 | DOI: 10.1016/j.neuroscience.2023.01.007

N-methyl-D-aspartate receptor (NMDAR) hypofunction during brain development is likely to contribute to the manifestation of schizophrenia (SCZ) in young adulthood. The cellular targets of NMDAR hypofunction appear to be at least in part corticolimbic fast-spiking (FS) interneurons. However, functional alterations in parvalbumin (PV)-positive FS interneurons following NMDAR hypofunction are poorly understood. Paired patch-clamp recordings from murine cortical PV interneurons and pyramidal neurons revealed that genetic deletion of NMDAR subunit Grin1 in prospective PV interneurons before the second postnatal week impaired evoked- and synchronized-GABA release. Whereas intrinsic excitability and spiking characteristics were also disturbed by Grin1 deletion, neither restoring their excitability by K+ channel blockade nor increasing extracellular Ca2+ rescued the GABA release. GABA release was also insensitive to the Cav2.1 channel antagonist ω-agatoxin IVA. Heterozygous deletion of Cacna1a gene (encoding Cav2.1) in PV interneurons produced a similar GABA release phenotype as the Grin1 mutants. Treatment with the Cav2.1/2.2 channel agonist GV-58 augmented somatic Ca2+ currents and GABA release in Cacna1a-haploinsufficient PV interneurons, but failed to enhance GABA release in the Grin1-deleted PV interneurons. Taken together, our results suggest that Grin1 deletion in prospective PV interneurons impairs proper maturation of membrane excitability and Cav2.1-recruited evoked GABA release. This may increase synaptic excitatory/inhibitory ratio in principal neurons, contributing to the emergence of SCZ-like phenotypes.

Chronic activation of the relaxin-3 receptor on GABA neurons in rat ventral hippocampus promotes anxiety and social avoidance

Hippocampus

2019 Mar 19

Rytova V, Ganella DE, Hawkes D, Bathgate RAD, Ma S and Gundlach AL
PMID: 30891856 | DOI: 10.1002/hipo.23089

Anxiety disorders are highly prevalent in modern society and better treatments are required. Key brain areas and signaling systems underlying anxiety include prefrontal cortex, hippocampus, and amygdala, and monoaminergic and peptidergic systems, respectively. Hindbrain GABAergic projection neurons that express the peptide, relaxin-3, broadly innervate the forebrain, particularly the septum and hippocampus, and relaxin-3 acts via a Gi/o -protein-coupled receptor known as the relaxin-family peptide 3 receptor (RXFP3). Thus, relaxin-3/RXFP3 signaling is implicated in modulation of arousal, motivation, mood, memory, and anxiety. Ventral hippocampus (vHip) is central to affective and cognitive processing and displays a high density of relaxin-3-positive nerve fibers and RXFP3 binding sites, but the identity of target neurons and associated effects on behavior are unknown. Therefore, in adult, male rats, we assessed the neurochemical nature of hippocampal RXFP3 mRNA-expressing neurons and anxiety-like and social behavior following chronic RXFP3 activation in vHip by viral vector expression of an RXFP3-selective agonist peptide, R3/I5. RXFP3 mRNA detected by fluorescent in situ hybridization was topographically distributed across the hippocampus in somatostatin- and parvalbumin-mRNA expressing GABA neurons. Chronic RXFP3 activation in vHip increased anxiety-like behavior in the light-dark box and elevated-plus maze, but not the large open-field test, and reduced social interaction with a conspecific stranger. Our data reveal disruptive effects of persistent RXFP3 signaling on hippocampal GABA networks important in anxiety; and identify a potential therapeutic target for anxiety disorders that warrants further investigation in relevant preclinical models.

Pathway-specific contribution of parvalbumin interneuron NMDARs to synaptic currents and thalamocortical feedforward inhibition

Molecular psychiatry

2022 Sep 08

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.

Muscarinic and NMDA receptors in the substantia nigra play a role in reward-related learning

The international journal of neuropsychopharmacology

2022 Nov 19

Galaj, E;Barrera, ED;Lynch, OL;Diodati, R;Thomas, A;Schneider, P;Lenhard, H;Vashisht, A;Ranaldi, R;
PMID: 36402549 | DOI: 10.1093/ijnp/pyac076

Reward-related learning, where animals form associations between rewards and stimuli (i.e., conditioned stimuli, CS) that predict or accompany those rewards, is an essential adaptive function for survival.In this study, we investigated the mechanisms underlying the acquisition and performance of conditioned approach learning with a focus on the role of muscarinic acetylcholine (mACh) and NMDA glutamate receptors in the substantia nigra (SN), a brain region implicated in reward and motor processes.Using RNAscope in situ hybridization assays, we found that dopamine neurons of the SN express muscarinic (mACh5), NMDA2a, NMDA2b, and NMDA2d receptor mRNA but not mACh4. NMDA, but not mACh5, receptor mRNA was also found on SN GABA neurons. In a conditioned approach paradigm, rats were exposed to 3 or 7 conditioning sessions during which light/tone (CS) presentations were paired with delivery of food pellets (US), followed by a test session with CS only presentations. Intra-SN microinjections of scopolamine (a mACh receptor antagonist) or AP-5 (a NMDA receptor antagonist) were made either prior to each conditioning session (to test their effects on acquisition) or prior to the CS-only test (to test their effects on expression of the learned response). Scopolamine and AP-5 produced dose-dependent significant reductions in the acquisition, but not performance, of conditioned approach.These results suggest that SN mACh and NMDA receptors are key players in the acquisition, but not the expression of reward-related learning. Importantly, these findings redefine the role of the SN, which has traditionally been known for its involvement in motor processes and suggest that the SN possesses attributes consistent with a function as a hub of integration of primary reward and conditioned stimulus signals.

Single-cell transcriptomics of the developing lateral geniculate nucleus reveals insights into circuit assembly and refinement

Proc Natl Acad Sci U S A.

2018 Jan 17

Kalish BT, Cheadle L, Hrvatin S, Nagy MA, Rivera S, Crow M, Gillis J, Kirchner R, Greenberg ME.
PMID: 29343640 | DOI: 10.1073/pnas.1717871115

Coordinated changes in gene expression underlie the early patterning and cell-type specification of the central nervous system. However, much less is known about how such changes contribute to later stages of circuit assembly and refinement. In this study, we employ single-cell RNA sequencing to develop a detailed, whole-transcriptome resource of gene expression across four time points in the developing dorsal lateral geniculate nucleus (LGN), a visual structure in the brain that undergoes a well-characterized program of postnatal circuit development. This approach identifies markers defining the major LGN cell types, including excitatory relay neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Most cell types exhibit significant transcriptional changes across development, dynamically expressing genes involved in distinct processes including retinotopic mapping, synaptogenesis, myelination, and synaptic refinement. Our data suggest that genes associated with synapse and circuit development are expressed in a larger proportion of nonneuronal cell types than previously appreciated. Furthermore, we used this single-cell expression atlas to identify the Prkcd-Cre mouse line as a tool for selective manipulation of relay neurons during a late stage of sensory-driven synaptic refinement. This transcriptomic resource provides a cellular map of gene expression across several cell types of the LGN, and offers insight into the molecular mechanisms of circuit development in the postnatal brain.

GABAergic interneurons expressing the α2 nicotinic receptor subunit are functionally integrated in the striatal microcircuit

Cell reports

2022 May 24

Tokarska, A;Silberberg, G;
PMID: 35613598 | DOI: 10.1016/j.celrep.2022.110842

The interactions between the striatal cholinergic and GABAergic systems are crucial in shaping reward-related behavior and reinforcement learning; however, the synaptic pathways mediating them are largely unknown. Here, we use Chrna2-Cre mice to characterize striatal interneurons (INs) expressing the nicotinic α2 receptor subunit. Using triple patch-clamp recordings combined with optogenetic stimulations, we characterize the electrophysiological, morphological, and synaptic properties of striatal Chrna2-INs. Striatal Chrna2-INs have diverse electrophysiological properties, distinct from their counterparts in other brain regions, including the hippocampus and neocortex. Unlike in other regions, most striatal Chrna2-INs are fast-spiking INs expressing parvalbumin. Striatal Chrna2-INs are intricately integrated in the striatal microcircuit, forming inhibitory synaptic connections with striatal projection neurons and INs, including other Chrna2-INs. They receive excitatory inputs from primary motor cortex mediated by both AMPA and NMDA receptors. A subpopulation of Chrna2-INs responds to nicotinic input, suggesting reciprocal interactions between this GABAergic interneuron population and striatal cholinergic synapses.

c-Maf-positive spinal cord neurons are critical elements of a dorsal horn circuit for mechanical hypersensitivity in neuropathy

Cell reports

2023 Mar 21

Frezel, N;Ranucci, M;Foster, E;Wende, H;Pelczar, P;Mendes, R;Ganley, RP;Werynska, K;d'Aquin, S;Beccarini, C;Birchmeier, C;Zeilhofer, HU;Wildner, H;
PMID: 36947543 | DOI: 10.1016/j.celrep.2023.112295

Corticospinal tract (CST) neurons innervate the deep spinal dorsal horn to sustain chronic neuropathic pain. The majority of neurons targeted by the CST are interneurons expressing the transcription factor c-Maf. Here, we used intersectional genetics to decipher the function of these neurons in dorsal horn sensory circuits. We find that excitatory c-Maf (c-MafEX) neurons receive sensory input mainly from myelinated fibers and target deep dorsal horn parabrachial projection neurons and superficial dorsal horn neurons, thereby connecting non-nociceptive input to nociceptive output structures. Silencing c-MafEX neurons has little effect in healthy mice but alleviates mechanical hypersensitivity in neuropathic mice. c-MafEX neurons also receive input from inhibitory c-Maf and parvalbumin neurons, and compromising inhibition by these neurons caused mechanical hypersensitivity and spontaneous aversive behaviors reminiscent of c-MafEX neuron activation. Our study identifies c-MafEX neurons as normally silent second-order nociceptors that become engaged in pathological pain signaling upon loss of inhibitory control.

Hippocampal and thalamic afferents form distinct synaptic microcircuits in the mouse infralimbic frontal cortex

Cell reports

2021 Oct 19

Graham, K;Spruston, N;Bloss, EB;
PMID: 34686328 | DOI: 10.1016/j.celrep.2021.109837

The selection of goal-directed behaviors is supported by neural circuits located within the frontal cortex. Frontal cortical afferents arise from multiple brain areas, yet the cell-type-specific targeting of these inputs is unclear. Here, we use monosynaptic retrograde rabies mapping to examine the distribution of afferent neurons targeting distinct classes of local inhibitory interneurons and excitatory projection neurons in mouse infralimbic frontal cortex. Interneurons expressing parvalbumin, somatostatin, or vasoactive intestinal peptide receive a large proportion of inputs from the hippocampus, while interneurons expressing neuron-derived neurotrophic factor receive a large proportion of inputs from thalamic regions. A similar dichotomy is present among the four different excitatory projection neurons. These results show a prominent bias among long-range hippocampal and thalamic afferent systems in their targeting to specific sets of frontal cortical neurons. Moreover, they suggest the presence of two distinct local microcircuits that control how different inputs govern frontal cortical information processing.

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sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
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XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
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UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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