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Background: Bipolar disorder is characterized by cyclical alternation between mania and depression, often comorbid with psychosis and suicide. Compared with other medications, the mood stabilizer lithium is the most effective treatment for the prevention of manic and depressive episodes. However, the pathophysiology of bipolar disorder and lithium’s mode of action are yet to be fully understood. Evidence suggests a change in the balance of excitatory and inhibitory activity, favouring excitation in bipolar disorder. In the present study, we sought to establish a holistic understanding of the neuronal consequences of lithium exposure in mouse cortical neurons, and to identify underlying mechanisms of action., Methods: We used a range of technical approaches to determine the effects of acute and chronic lithium treatment on mature mouse cortical neurons. We combined RNA screening and biochemical and electrophysiological approaches with confocal immunofluorescence and live-cell calcium imaging., Results: We found that only chronic lithium treatment significantly reduced intracellular calcium flux, specifically by activating metabotropic glutamatergic receptor 5. This was associated with altered phosphorylation of protein kinase C and glycogen synthase kinase 3, reduced neuronal excitability and several alterations to synapse function. Consequently, lithium treatment shifts the excitatory–inhibitory balance toward inhibition., Limitations: The mechanisms we identified should be validated in future by similar experiments in whole animals and human neurons., Conclusion: Together, the results revealed how lithium dampens neuronal excitability and the activity of the glutamatergic network, both of which are predicted to be overactive in the manic phase of bipolar disorder. Our working model of lithium action enables the development of targeted strategies to restore the balance of overactive networks, mimicking the therapeutic benefits of lithium but with reduced toxicity., Competing Interests: None declared., (© 2021 CMA Joule Inc. or its licensors)

Leucine-rich glioma-inactivated protein 1 (LGI1) is a secreted neuronal protein and a Nogo receptor 1 (NgR1) ligand. Mutations in LGI1 in humans causes autosomal dominant lateral temporal lobe epilepsy and homozygous deletion of LGI1 in mice results in severe epileptic seizures that cause early postnatal death. NgR1 plays an important role in the development of CNS synapses and circuitry by limiting plasticity in the adult cortex via the activation of RhoA. These relationships and functions prompted us to examine the effect of LGI1 on synapse formation in vitro and in vivo . We report that application of LGI1 increases synaptic density in neuronal culture and that LGI1 null hippocampus has fewer dendritic mushroom spines than in wild-type (WT) littermates. Further, our electrophysiological investigations demonstrate that LGI1 null hippocampal neurons possess fewer and weaker synapses. RhoA activity is significantly increased in cortical cultures derived from LGI1 null mice and using a reconstituted system; we show directly that LGI1 antagonizes NgR1-tumor necrosis factor receptor orphan Y (TROY) signaling. Our data suggests that LGI1 enhances synapse formation in cortical and hippocampal neurons by reducing NgR1 signaling.

Persistent firing of entorhinal cortex (EC) pyramidal neurons is a key component of working and spatial memory. We report here that a pro-brain-derived neurotrophic factor (proBDNF)-dependent p75NTR signaling pathway plays a major role in excitability and persistent activity of pyramidal neurons in layer V of the EC. Using electrophysiological recordings, we show that proBDNF suppresses persistent firing in entorhinal slices from wild-type mice but not from p75NTR-null mice. Conversely, function-blocking proBDNF antibodies enhance excitability of pyramidal neurons and facilitate their persistent firing, and acute exposure to function-blocking p75NTR antibodies results in enhanced firing activity of pyramidal neurons. Genetic deletion of p75NTR specifically in neurons or during adulthood also induces enhanced excitability and persistent activity, indicating that the proBDNF-p75NTR signaling cascade functions within adult neurons to inhibit pyramidal activity. Phosphatidylinositol 4,5-bisphosphate (PIP2)-sensitive transient receptor potential canonical channels play a critical role in mediating persistent firing in the EC and we hypothesized that proBDNF-dependent p75NTR activation regulates PIP2 levels. Accordingly, proBDNF decreases cholinergic calcium responses in cortical neurons and affects carbachol-induced depletion of PIP2. Further, we show that the modulation of persistent firing by proBDNF relies on a p75NTR-Rac1-PI4K pathway. The hypothesis that proBDNF and p75NTR maintain network homeostasis in the adult CNS was tested in vivo and we report that p75NTR-null mice show improvements in working memory but also display an increased propensity for severe seizures. We propose that the proBDNF-p75NTR axis controls pyramidal neuron excitability and persistent activity to balance EC performance with the risk of runaway activity., Significance Statement: Persistent firing of entorhinal cortex (EC) pyramidal neurons is required for working memory. We report here that pro-brain-derived neurotrophic factor (proBDNF) activates p75NTR to induce a Rac1-dependent and phosphatidylinositol 4,5-bisphosphate-dependent signaling cascade that suppresses persistent activity. Conversely, using loss-of-function approaches, we find that endogenous proBDNF or p75NTR activation strongly decreases pyramidal neuron excitability and persistent firing, suggesting that a physiological role of this proBDNF-p75NTR cascade may be to regulate working memory in vivo. Consistent with this, mice rendered null for p75NTR during adulthood show improvements in working memory but also display an increased propensity for severe seizures. We propose that by attenuating EC network performance, the proBDNF-p75NTR signaling cascade reduces the probability of epileptogenesis., (Copyright © 2015 the authors 0270-6474/15/359741-13$15.00/0.)

Functional cross-talk between structurally unrelated P2X ATP receptors and members of the 'cys-loop' receptor-channel superfamily represents a recently-discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross-talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross-inhibition between P2X subtypes and GABA(A) receptors, suggesting a P2X subunit-dependent mechanism. We show here that cross-inhibition between neuronal P2X(3) or P2X(2+3) and GABA(A) receptors does not depend on chloride and calcium ions. We identified an intracellular QST(386-388) motif in P2X(3) subunits which is required for the functional coupling with GABA(A) receptors. Moreover the cross-inhibition between native P2X(3) and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABA(A)beta3 subunits. We provide evidence that P2X(3) and GABA(A) receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X(3)-GABA(A) subunit interactions underlie a pre-synaptic cross-talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.

During muscarinic modulation, principal neurons from layer V of rat medial entorhinal cortex (mEC) respond to repeated applications of a brief stimulus with a graded change in persistent firing frequency. This pattern of discharge has been proposed to represent an intrinsic mechanism for short-term memory operations. To investigate the implementation of persistent activity in mEC during development, we characterized the electrophysiological properties of layer V principal neurons in the mEC over a range of postnatal stages. We observed significant differences in both passive (resistance, time constant, and resting membrane potential) and active properties (threshold, action potential, and adaptation) of principal neurons from rats aged 5-7, 10-13, 16-19, and 21-23 days. We also examined the properties of muscarinic-dependent persistent activity in EC slices from different age groups. Recordings were conducted using the perforated-patch whole cell technique because persistent activity runs down in the ruptured-patch configuration. Although no neuron in the youngest group exhibited graded persistent activity in response to muscarinic receptor activation, this activity was recorded in the 10- to 13-day-old group and its occurrence increased from 69% in the 16- to 19-day-old group to 76% in the 21- to 23-day-old group. This postnatal increase in neurons endowed with persistent firing properties in mEC was found to parallel the increase in density of ChAT-positive immunostaining of fibers and the developmental changes in M1 muscarinic receptor mRNA levels. All these data suggest that the implementation of mnemonic properties in mEC principal neurons matches the ontogenic development of afferent cholinergic circuits and their signaling components.

Body fluid homeostasis requires the release of arginine-vasopressin (AVP, an antidiuretic hormone) from the neurohypophysis. This release is controlled by specific and highly sensitive 'osmoreceptors' in the hypothalamus. Indeed, AVP-releasing neurons in the supraoptic nucleus (SON) are directly osmosensitive, and this osmosensitivity is mediated by stretch-inhibited cation channels. However, the molecular nature of these channels remains unknown. Here we show that SON neurons express an N-terminal splice variant of the transient receptor potential vanilloid type-1 (Trpv1), also known as the capsaicin receptor, but not full-length Trpv1. Unlike their wild-type counterparts, SON neurons in Trpv1 knockout (Trpv1(-/-)) mice could not generate ruthenium red-sensitive increases in membrane conductance and depolarizing potentials in response to hyperosmotic stimulation. Moreover, Trpv1(-/-) mice showed a pronounced serum hyperosmolality under basal conditions and severely compromised AVP responses to osmotic stimulation in vivo. These results suggest that the Trpv1 gene may encode a central component of the osmoreceptor.

Molecular and behavioral evidence suggests that acid-sensing ion channels (ASICs) contribute to pain processing, but an understanding of their precise role remains elusive. Existing ASIC knock-out mouse experiments are complicated by the heteromultimerization of ASIC subunits. Therefore, we have generated transgenic mice that express a dominant-negative form of the ASIC3 subunit that inactivates all native neuronal ASIC-like currents by oligomerization. Using whole-cell patch-clamp recordings, we examined the response properties of acutely isolated dorsal root ganglion neurons to protons (pH 5.0). We found that whereas 33% of the proton-responsive neurons from wild-type mice exhibited an ASIC-like transient response, none of the neurons from the transgenic mice exhibited a transient inward current. Capsaicin-evoked responses mediated by the TRPV1 receptor were unaltered in transgenic mice. Adult male wild-type and transgenic mice were subjected to a battery of behavioral nociceptive assays, including tests of thermal, mechanical, chemical/inflammatory, and muscle pain. The two genotypes were equally sensitive to thermal pain and to thermal hypersensitivity after inflammation. Compared with wild types, however, transgenic mice were more sensitive to a number of modalities, including mechanical pain (von Frey test, tail-clip test), chemical/inflammatory pain (formalin test, 0.6% acetic acid writhing test), mechanical hypersensitivity after zymosan inflammation, and mechanical hypersensitivity after intramuscular injection of hypotonic saline. These data reinforce the hypothesis that ASICs are involved in both mechanical and inflammatory pain, although the increased sensitivity of transgenic mice renders it unlikely that they are direct transducers of nociceptive stimuli.

P2X receptors have been suggested to be expressed on the central terminals of A delta-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X2+3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X1, P2X3 homomers, and P2X2+3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined alpha,beta-methylene-ATP (alpha beta meATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. alpha beta meATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 microM TNP-ATP (n = 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 microM TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 microM alpha beta meATP could be largely inhibited by 10 microM iso-pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive A delta-afferent fibers and play a role in modulating sensory transmission to lamina V neurons.

Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X2 ATP-gated channels and 5-HT3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X2 and 5-HT3 receptors is disrupted when the intracellular C-terminal domain of the P2X2 receptor subunit is deleted and when minigenes coding for P2X2 or 5-HT3A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X2 receptors in myenteric neurons also disrupts the functional interaction between native P2X2 and 5-HT3 receptors. Therefore, activity-dependent intracellular coupling of distinct receptor channels underlies ionotropic cross talks that may significantly contribute to the regulation of neuronal excitability and synaptic plasticity.

The distribution and morphological characteristics of dopamine (DA) neurons in the midbrain of the squirrel monkey (Saimiri sciureus) were investigated by peroxidase-antiperoxidase (PAP) immunohistochemistry with a highly specific antiserum raised against DA-glutaraldehyde-lysyl-protein conjugate (donated by M. Geffard). Four contiguous areas contained DA-immunostained nerve cell bodies: (1) the substantia nigra, pars compacta (SNc), (2) the ventral tegmental area (VTA), (3) the retrorubral area (RRA), and (4) the periaqueductal gray (PAG). The SNc composed the vast majority of DA-immunostained neurons. Most of these neurons were relatively large (mean diameters: 35 x 15 micron) and varied in shape from fusiform to polygonal, but a few smaller (16 x 10.5 micron) globular cells were dispersed among them. The caudal two-thirds of the SNc was particularly rich in DA somata. Rostrally, these DA cells formed several distinct columns impinging deeply upon the underlying pars reticulata. Large oval sectors mostly devoid of immunoreactivity were delineated by these trabeculae. The long dendritic processes of DA neurons in the SNc were generally oriented in prominent dorsoventral bundles the ventralmost portion of which arborized diffusely along the dorsal surface of the cerebral peduncle. In the VTA, the DA neurons were regrouped in a triangular zone located dorsal to the interpeduncular nucleus, medial to the substantia nigra and ventral to the oculomotor nucleus. These DA cells were of medium size (19 x 10.5 micron), globular or fusiform, and usually showed one or two thick primary dendrites oriented dorsoventrally. The DA cells in the RRA lay in continuity with the most caudal DA-containing elements of the substantia nigra but could be distinguished by their smaller size (26 x 12 micron), shorter and more profusely branched dendrites, and darker immunostaining. These DA neurons were characteristically scattered among and medial to the fibers of the medial lemniscus, and a few could be observed as far caudally as the pedunculopontine nucleus. In the PAG, DA-immunostained neurons were seen in the rostral half of the mesencephalic central gray and predominated in its ventral half. These cells were of medium size (22.5 x 10 micron) and some of them were found in proximity to the ventricular lining. At caudal levels, the DA-positive cells in the PAG did not intermingle with dorsal raphe neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

A light microscopic study of the cellular localization of GABA in the thalamus of the squirrel monkey (Saimiri sciureus) was undertaken by means of the indirect peroxidase-antiperoxidase method using a highly purified antiserum directed against GABA-glutaraldehyde-lysyl-protein conjugate. GABA-immunoreactive cell bodies and axon terminals were visualized in all thalamic nuclei in the squirrel monkey but their relative density varied from one nucleus to the other. At the level of the anterior nuclear group, GABA-positive cells and terminals abounded in the anterodorsal nucleus but were much less numerous in the anteromedial and anteroventral nuclei. In the nuclei of the ventral group, GABA-immunoreactive cells were found to be smaller and less numerous than nonimmunoreactive neurons. In the ventral anterior nucleus, GABA-positive neuronal profiles formed typical clusters, whereas they were more uniformly distributed in the posterior nuclei of the ventral group. In the intralaminar nuclei, GABA-immunoreactive cells and terminals abounded in the dorsal portion of the paracentral and centrolateral nuclei, whereas more caudally, GABA-positive terminals pervaded the entire parafascicular nucleus. In the mediodorsal nucleus, GABA-positive cell bodies and axon terminals formed typical clusters of various sizes scattered within the lateral parvocellular portion of the nucleus, while GABA-immunoreactive neuronal profiles were less numerous and more uniformly distributed in the medial portion of this structure. In the nuclei of the posterior group, GABA-immunoreactive neuronal profiles were uniformly distributed except in the pulvinar where they abounded in the inferior and oral parts but were scarce in the medial part. In the dorsal lateral geniculate nucleus, the magnocellular layers received the most massive GABA-positive innervation and contained the largest number of GABA-immunoreactive cell bodies. In the ventral lateral geniculate nucleus, GABA-positive cells occurred only ventrolaterally while GABA-immunoreactive terminals pervaded the entire structure. In the medial geniculate nucleus, GABA-immunoreactive cell bodies and terminals abounded particularly within the ventromedial third of the structure. In the habenula, a few GABA-immunoreactive cell bodies and numerous GABA-positive terminals were scattered throughout the lateral habenular nucleus, whereas only a few GABA-immunoreactive terminals surrounded the closely packed unreactive cells in the medial habenular nucleus. In contrast to other thalamic nuclei all neurons in the reticular nucleus displayed GABA immunoreactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

AbstractThe anterior cingulate cortex (ACC) processes the affective component of pain, whereas the primary somatosensory cortex (S1) is involved in its sensory-discriminative component. Injection of morphine in the ACC has been reported to be analgesic, and endogenous opioids in this area are required for pain relief. Mu opioid receptors (MORs) are expressed in both ACC and S1; however, the identity of MOR-expressing cortical neurons remains unknown. Using the Oprm1-mCherry mouse line, we performed selective patch clamp recordings of MOR+ neurons, as well as immunohistochemistry with validated neuronal markers, to determine the identity and laminar distribution of MOR+ neurons in ACC and S1. We found that the electrophysiological signatures of MOR+ neurons differ significantly between these 2 areas, with interneuron-like firing patterns more frequent in ACC. While MOR+ somatostatin interneurons are more prominent in ACC, MOR+ excitatory neurons and MOR+ parvalbumin interneurons are more prominent in S1. Our results suggest a differential contribution of MOR-mediated modulation to ACC and S1 outputs. We also found that females had a greater density of MOR+ neurons compared with males in both areas. In summary, we conclude that MOR-dependent opioidergic signaling in the cortex displays sexual dimorphisms and likely evolved to meet the distinct function of pain-processing circuits in limbic and sensory cortical areas.