Impaired Lactate Release in Dorsal CA1 Astrocytes Contributed to Nociceptive Sensitization and Comorbid Memory Deficits in Rodents.

BACKGROUND: Memory deficits are a common comorbid disorder in patients suffering from neuropathic pain. The mechanisms underlying the comorbidities remain elusive. The hypothesis of this study was that impaired lactate release from dysfunctional astrocytes in dorsal hippocampal CA1 contributed to memory deficits. METHODS: A spared nerve injury model was established to induce both pain and memory deficits in rats and mice of both sexes. von Frey tests, novel object recognition, and conditioned place preference tests were applied to evaluate the behaviors. Whole-cell recording, fiber photometry, Western blotting, and immunohistochemistry combined with intracranial injections were used to explore the underlying mechanisms. RESULTS: Animals with spared sciatic nerve injury that had displayed nociception sensitization or memory deficit comorbidities demonstrated a reduction in the intrinsic excitability of pyramidal neurons, accompanied by reduced Ca2+ activation in astrocytes (ΔF/F, sham: 6 ± 2%; comorbidity: 2 ± 0.4%) and a decrease in the expression of glial fibrillary acidic protein and lactate levels in the dorsal CA1. Exogenous lactate supply or increasing endogenous lactate release by chemogenetic activation of astrocytes alleviated this comorbidity by enhancing the cell excitability (129 ± 4 vs. 88 ± 10 for 3.5 mM lactate) and potentiating N-methyl-d-aspartate receptor-mediated excitatory postsynaptic potentials of pyramidal neurons. In contrast, inhibition of lactate synthesis, blocking lactate transporters, or chemogenetic inhibition of astrocytes resulted in comorbidity-like behaviors in naive animals. Notably, ß2-adrenergic receptors in astrocytes but not neurons were downregulated in dorsal CA1 after spared nerve injury. Microinjection of a ß2 receptor agonist into dorsal CA1 or activation of the noradrenergic projections onto the hippocampus from the locus coeruleus alleviated the comorbidity, possibly by increasing lactate release. CONCLUSIONS: Impaired lactate release from dysfunctional astrocytes, which could be rescued by activation of the locus coeruleus, led to nociception and memory deficits after peripheral nerve injury.

IPAC integrates rewarding and environmental memory during the acquisition of morphine CPP.

The association between rewarding and drug-related memory is a leading factor for the formation of addiction, yet the neural circuits underlying the association remain unclear. Here, we showed that the interstitial nucleus of the posterior limb of the anterior commissure (IPAC) integrated rewarding and environmental memory information by two different receiving projections from ventral tegmental area (VTA) and nucleus accumbens shell region (NAcSh) to mediate the acquisition of morphine conditioned place preference (CPP). A projection from the VTA GABAergic neurons (VTAGABA) to the IPAC lateral region GABAergic neurons (IPACLGABA) mediated the effect of morphine rewarding, whereas the pathway from NAcSh dopamine receptor 1-expressing neurons (NAcShD1) to the IPAC medial region GABAergic neurons (IPACMGABA) was involved in the acquisition of environmental memory. These findings demonstrated that the distinct IPAC circuits VTAGABA→IPACLGABA and NAcShD1R→IPACMGABA were attributable to the rewarding and environmental memory during the acquisition of morphine CPP, respectively, and provided the circuit-based potential targets for preventing and treating opioid addiction.

Tail nerve electrical stimulation promoted the efficiency of transplanted spinal cord-like tissue as a neuronal relay to repair the motor function of rats with transected spinal cord injury.

Following transected spinal cord injury (SCI), there is a critical need to restore nerve conduction at the injury site and activate the silent neural circuits caudal to the injury to promote the recovery of voluntary movement. In this study, we generated a rat model of SCI, constructed neural stem cell (NSC)-derived spinal cord-like tissue (SCLT), and evaluated its ability to replace injured spinal cord and repair nerve conduction in the spinal cord as a neuronal relay. The lumbosacral spinal cord was further activated with tail nerve electrical stimulation (TNES) as a synergistic electrical stimulation to better receive the neural information transmitted by the SCLT. Next, we investigated the neuromodulatory mechanism underlying the action of TNES and its synergism with SCLT in SCI repair. TNES promoted the regeneration and remyelination of axons and increased the proportion of glutamatergic neurons in SCLT to transmit brain-derived neural information more efficiently to the caudal spinal cord. TNES also increased the innervation of motor neurons to hindlimb muscle and improved the microenvironment of muscle tissue, resulting in effective prevention of hindlimb muscle atrophy and enhanced muscle mitochondrial energy metabolism. Tracing of the neural circuits of the sciatic nerve and tail nerve identified the mechanisms responsible for the synergistic effects of SCLT transplantation and TNES in activating central pattern generator (CPG) neural circuits and promoting voluntary motor function recovery in rats. The combination of SCLT and TNES is expected to provide a new breakthrough for patients with SCI to restore voluntary movement and control their muscles.

Corticotropin releasing factor neurons in the visual cortex mediate long-term changes in visual function induced by early adversity.

Early adversity can cause malfunction of the visual system in adulthood. Adult female but not male mice undergoing early chronic mild stress (ECMS) maintain ocular dominance (OD) plasticity after the critical period. How early stressful experiences have a long-term impact on it is largely unknown. Here, we observed a wide distribution of corticotropin-releasing factor (CRF)-positive neurons, which mainly colocalized with a subpopulation of GABAergic interneurons in the mouse primary visual cortex (V1). Optogenetic activation of CRF-positive neurons transfected with AAV-ChR2 evoked inhibitory currents in nearby pyramidal cells. ECMS induced a reduction in the expression of CRF mRNA in adult mouse V1. Chemogenetic activation of V1 CRF neurons impaired OD plasticity in adult ECMS females. We further showed that local administration of the corticotropin releasing factor receptor 1 (CRFR1) antagonist via an osmotic minipump into the visual cortex mimicked OD plasticity in adult ECMS females. Whole-cell recording in layer 2/3 pyramidal neurons revealed that the CRFR1 antagonist reduced the short-term depression (STD) of evoked inhibitory postsynaptic current (IPSC) in females but not in males. Likewise, CRF agonists have the opposite effect. In summary, our findings indicate that the local CRF-CRFR1 system within V1 may mediate the long-term and sex-dependent effect of early stress experiences on visual plasticity and provide a target for the prevention of visual deficits in adults with a history of early-life adversity.

Celsr2 regulates NMDA receptors and dendritic homeostasis in dorsal CA1 to enable social memory.

Social recognition and memory are critical for survival. The hippocampus serves as a central neural substrate underlying the dynamic coding and transmission of social information. Yet the molecular mechanisms regulating social memory integrity in hippocampus remain unelucidated. Here we report unexpected roles of Celsr2, an atypical cadherin, in regulating hippocampal synaptic plasticity and social memory in mice. Celsr2-deficient mice exhibited defective social memory, with rather intact levels of sociability. In vivo fiber photometry recordings disclosed decreased neural activity of dorsal CA1 pyramidal neuron in Celsr2 mutants performing social memory task. Celsr2 deficiency led to selective impairment in NMDAR but not AMPAR-mediated synaptic transmission, and to neuronal hypoactivity in dorsal CA1. Those activity changes were accompanied with exuberant apical dendrites and immaturity of spines of CA1 pyramidal neurons. Strikingly, knockdown of Celsr2 in adult hippocampus recapitulated the behavioral and cellular changes observed in knockout mice. Restoring NMDAR transmission or CA1 neuronal activities rescued social memory deficits. Collectively, these results show a critical role of Celsr2 in orchestrating dorsal hippocampal NMDAR function, dendritic and spine homeostasis, and social memory in adulthood.

Electroacupuncture facilitates the integration of a grafted TrkC-modified mesenchymal stem cell-derived neural network into transected spinal cord in rats via increasing neurotrophin-3.

AIMS: This study was aimed to investigate whether electroacupuncture (EA) would increase the secretion of neurotrophin-3 (NT-3) from injured spinal cord tissue, and, if so, whether the increased NT-3 would promote the survival, differentiation, and migration of grafted tyrosine kinase C (TrkC)-modified mesenchymal stem cell (MSC)-derived neural network cells. We next sought to determine if the latter would integrate with the host spinal cord neural circuit to improve the neurological function of injured spinal cord. METHODS: After NT-3-modified Schwann cells (SCs) and TrkC-modified MSCs were co-cultured in a gelatin sponge scaffold for 14 days, the MSCs differentiated into neuron-like cells that formed a MSC-derived neural network (MN) implant. On this basis, we combined the MN implantation with EA in a rat model of spinal cord injury (SCI) and performed immunohistochemical staining, neural tracing, electrophysiology, and behavioral testing after 8 weeks. RESULTS: Electroacupuncture application enhanced the production of endogenous NT-3 in damaged spinal cord tissues. The increase in local NT-3 production promoted the survival, migration, and maintenance of the grafted MN, which expressed NT-3 high-affinity TrkC. The combination of MN implantation and EA application improved cortical motor-evoked potential relay and facilitated the locomotor performance of the paralyzed hindlimb compared with those of controls. These results suggest that the MN was better integrated into the host spinal cord neural network after EA treatment compared with control treatment. CONCLUSIONS: Electroacupuncture as an adjuvant therapy for TrkC-modified MSC-derived MN, acted by increasing the local production of NT-3, which accelerated neural network reconstruction and restoration of spinal cord function following SCI.

Autism-like social deficit generated by Dock4 deficiency is rescued by restoration of Rac1 activity and NMDA receptor function.

Genetic studies of autism spectrum disorder (ASD) have revealed multigene variations that converge on synaptic dysfunction. DOCK4, a gene at 7q31.1 that encodes the Rac1 guanine nucleotide exchange factor Dock4, has been identified as a risk gene for ASD and other neuropsychiatric disorders. However, whether and how Dock4 disruption leads to ASD features through a synaptic mechanism remain unexplored. We generated and characterized a line of Dock4 knockout (KO) mice, which intriguingly displayed a series of ASD-like behaviors, including impaired social novelty preference, abnormal isolation-induced pup vocalizations, elevated anxiety, and perturbed object and spatial learning. Mice with conditional deletion of Dock4 in hippocampal CA1 recapitulated social preference deficit in KO mice. Examination in CA1 pyramidal neurons revealed that excitatory synaptic transmission was drastically attenuated in KO mice, accompanied by decreased spine density and synaptic content of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)- and NMDA (N-methyl-D-aspartate)-type glutamate receptors. Moreover, Dock4 deficiency markedly reduced Rac1 activity in the hippocampus, which resulted in downregulation of global protein synthesis and diminished expression of AMPA and NMDA receptor subunits. Notably, Rac1 replenishment in the hippocampal CA1 of Dock4 KO mice restored excitatory synaptic transmission and corrected impaired social deficits in these mice, and pharmacological activation of NMDA receptors also restored social novelty preference in Dock4 KO mice. Together, our findings uncover a previously unrecognized Dock4-Rac1-dependent mechanism involved in regulating hippocampal excitatory synaptic transmission and social behavior.

Tissue-Engineered Neural Network Graft Relays Excitatory Signal in the Completely Transected Canine Spinal Cord.

Tissue engineering produces constructs with defined functions for the targeted treatment of damaged tissue. A complete spinal cord injury (SCI) model is generated in canines to test whether in vitro constructed neural network (NN) tissues can relay the excitatory signal across the lesion gap to the caudal spinal cord. Established protocols are used to construct neural stem cell (NSC)-derived NN tissue characterized by a predominantly neuronal population with robust trans-synaptic activities and myelination. The NN tissue is implanted into the gap immediately following complete transection SCI of canines at the T10 spinal cord segment. The data show significant motor recovery of paralyzed pelvic limbs, as evaluated by Olby scoring and cortical motor evoked potential (CMEP) detection. The NN tissue survives in the lesion area with neuronal phenotype maintenance, improves descending and ascending nerve fiber regeneration, and synaptic integration with host neural circuits that allow it to serve as a neuronal relay to transmit excitatory electrical signal across the injured area to the caudal spinal cord. These results suggest that tissue-engineered NN grafts can relay the excitatory signal in the completely transected canine spinal cord, providing a promising strategy for SCI treatment in large animals, including humans.

Electroacupuncture Facilitates the Integration of Neural Stem Cell-Derived Neural Network with Transected Rat Spinal Cord.

The hostile environment of an injured spinal cord makes it challenging to achieve higher viability in a grafted tissue-engineered neural network used to reconstruct the spinal cord circuit. Here, we investigate whether cell survival and synaptic transmission within an NT-3 and TRKC gene-overexpressing neural stem cell-derived neural network scaffold (NN) transplanted into transected spinal cord could be promoted by electroacupuncture (EA) through improving the microenvironment. Our results showed that EA facilitated the cell survival, neuronal differentiation, and synapse formation of a transplanted NN. Pseudorabies virus tracing demonstrated that EA strengthened synaptic integration of the transplanted NN with the host neural circuit. The combination therapy also promoted axonal regeneration, spinal conductivity, and functional recovery. The findings highlight EA as a potential and safe supplementary therapeutic strategy to reinforce the survival and synaptogenesis of a transplanted NN as a neuronal relay to bridge the two severed ends of an injured spinal cord.

A Modular Assembly of Spinal Cord-Like Tissue Allows Targeted Tissue Repair in the Transected Spinal Cord.

Tissue engineering-based neural construction holds promise in providing organoids with defined differentiation and therapeutic potentials. Here, a bioengineered transplantable spinal cord-like tissue (SCLT) is assembled in vitro by simulating the white matter and gray matter composition of the spinal cord using neural stem cell-based tissue engineering technique. Whether the organoid would execute targeted repair in injured spinal cord is evaluated. The integrated SCLT, assembled by white matter-like tissue (WMLT) module and gray matter-like tissue (GMLT) module, shares architectural, phenotypic, and functional similarities to the adult rat spinal cord. Organotypic coculturing with the dorsal root ganglion or muscle cells shows that the SCLT embraces spinal cord organogenesis potentials to establish connections with the targets, respectively. Transplantation of the SCLT into the transected spinal cord results in a significant motor function recovery of the paralyzed hind limbs in rats. Additionally, targeted spinal cord tissue repair is achieved by the modular design of SCLT, as evidenced by an increased remyelination in the WMLT area and an enlarged innervation in the GMLT area. More importantly, the pro-regeneration milieu facilitates the formation of a neuronal relay by the donor neurons, allowing the conduction of descending and ascending neural inputs.

Recovery of paralyzed limb motor function in canine with complete spinal cord injury following implantation of MSC-derived neural network tissue.

We have reported previously that bone marrow mesenchymal stem cell (MSC)-derived neural network scaffold not only survived in the injury/graft site of spinal cord but also served as a "neuronal relay" that was capable of improving the limb motor function in a complete spinal cord injury (SCI) rat model. It remained to be explored whether such a strategy was effective for repairing the large spinal cord tissue loss as well as restoring motor function in larger animals. We have therefore extended in this study to construct a canine MSC-derived neural network tissue in vitro with the aim to evaluate its efficacy in treating adult beagle dog subjected to a complete transection of the spinal cord. The results showed that after co-culturing with neurotropin-3 overexpressing Schwann cells in a gelatin sponge scaffold for 14 days, TrkC overexpressing MSCs differentiated into neuron-like cells. In the latter, some cells appeared to make contacts with each other through synapse-like structures with trans-synaptic electrical activities. Remarkably, the SCI canines receiving the transplantation of the MSC-derived neural network tissue demonstrated a gradual restoration of paralyzed limb motor function, along with improved electrophysiological presentation when compared with the control group. Magnetic resonance imaging and diffusion tensor imaging showed that the canines receiving the MSC-derived neural network tissue exhibited robust nerve tract regeneration in the injury/graft site. Histological analysis showed that some of the MSC-derived neuron-like cells had survived in the injury/graft site up to 6.5 months. Implantation of MSC-derived neural network tissue significantly improved the microenvironment of the injury/graft site. It is noteworthy that a variable number of them had integrated with the regenerating corticospinal tract nerve fibers and 5-HT nerve fibers through formation of synapse-like contacts. The results suggest that the transplanted MSC-derived neural network tissue may serve as a structural and functional "neuronal relay" to restore the paralyzed limb motor function in the canine with complete SCI.

Inhibition of Cdk5 rejuvenates inhibitory circuits and restores experience-dependent plasticity in adult visual cortex.

Cyclin-dependent kinase 5 (Cdk5) acts as an essential modulator for neural development and neurological disorders. Here we show that Cdk5 plays a pivotal role in modulating GABAergic signaling and the maturation of visual system. In adult mouse primary visual cortex, Cdk5 formed complex with the GABA synthetic enzyme glutamate decarboxylase GAD67, but not with GAD65. In addition to enhancement in the surface level of NR2B-containing NMDA receptors, inhibition of Cdk5 reduced the protein levels of GADs and Otx2, while leaving intact the expression of vesicular GABA transporter and subunits of GABAA or AMPA receptors. Whole-cell patch-clamp recording in layer II/III pyramidal neurons revealed a decrease in the frequency of miniature inhibitory postsynaptic current (mIPSC). Consequently, pharmacological inhibition and genetic knockdown of Cdk5 in adult mice led to a restoration of juvenile-like ocular dominance plasticity in vivo and long-term synaptic potential in layer II/III induced by white matter stimulation in vitro. Interestingly, we did not observe an alteration of perineuronal nets of extracellular matrix, but a reinstatement of the capability to evoke long-term depression at inhibitory synapses (iLTD), which depended on presynaptic endocannabinoid receptors and was a sign of the rejuvenated GABAergic synapses. Enhancement of GABA signaling by diazepam impeded ocular dominance plasticity rescued by Cdk5 inhibition. These results thus suggest that a physiological role of Cdk5 in visual cortex is to consolidate and stabilize neural circuits through controlling GABAergic signaling.

Brief Novel Visual Experience Fundamentally Changes Synaptic Plasticity in the Mouse Visual Cortex.

LTP has been known to be a mechanism by which experience modifies synaptic responses in the neocortex. Visual deprivation in the form of dark exposure or dark rearing from birth enhances NMDAR-dependent LTP in layer 2/3 of visual cortex, a process often termed metaplasticity, which may involve changes in NMDAR subunit composition and function. However, the effects of reexposure to light after dark rearing from birth on LTP induction have not been explored. Here, we showed that the light exposure after dark rearing revealed a novel NMDAR independent form of LTP in the layer 2/3 pyramidal cells in visual cortex of mice of both sexes, which is dependent on mGluR5 activation and is associated with intracellular Ca2+ rise, CaMKII activity, PKC activity, and intact protein synthesis. Moreover, the capacity to induce mGluR-dependent LTP is transient: it only occurs when mice of both sexes reared in the dark from birth are exposed to light for 10-12 h, and it does not occur in vision-experienced, male mice, even after prolonged exposure to dark. Thus, the mGluR5-LTP unmasked by short visual experience can only be observed after dark rearing but not after dark exposure. These results suggested that, as in hippocampus, in layer 2/3 of visual cortex, there is coexistence of two distinct activity-dependent systems of synaptic plasticity, NMDAR-LTP, and mGluR5-LTP. The mGluR5-LTP unmasked by short visual experience may play a critical role in the faster establishment of normal receptive field properties.SIGNIFICANCE STATEMENT LTP has been known to be a mechanism by which experience modifies synaptic responses in the neocortex. Visual deprivation in the form of dark exposure or dark rearing from birth enhances NMDAR-dependent LTP in layer 2/3 of visual cortex, a process often termed metaplasticity. NMDAR-dependent form of LTP in visual cortex has been well characterized. Here, we report that an NMDAR-independent form of LTP can be promoted by novel visual experience on dark-reared mice, characterized as dependent on intracellular Ca2+ rise, PKC activity, and intact protein synthesis and also requires the activation of mGluR5. These findings suggest that, in layer 2/3 of visual cortex, as in hippocampus, there is coexistence of two distinct activity-dependent systems of synaptic plasticity.

Layer-specific endocannabinoid-mediated long-term depression of GABAergic neurotransmission onto principal neurons in mouse visual cortex.

Visually induced endocannabinoid-mediated long-term depression of GABAergic neurotransmission (iLTD) mediates the maturation of GABAergic release in layer 2/3 of visual cortex. Here we examined whether the maturation of GABAergic transmission in other layers of visual cortex also requires endocannabinoids. The developmental plasticity of GABAergic neurotransmission onto the principal neurons in different layers of mouse visual cortex was examined in cortical slices by whole-cell recordings of inhibitory postsynaptic currents evoked by presynaptic inhibitory inputs. Theta burst stimulation of GABAergic inputs induced an endocannabinoid-mediated long-term depression of GABAergic neurotransmission onto pyramidal cells in layer 2/3 from postnatal day (P)10 to 30 and in layer 5 from P10 to 40, whereas that of GABAergic inputs did not induce iLTD onto star pyramidal neurons in layer 4 at any time postnatally, indicating that this plasticity is laminar-specific. The developmental loss of iLTD paralleled the maturation of GABAergic inhibition in both layer 2/3 and layer 5. Visual deprivation delayed the developmental loss of iLTD in layers 3 and 5 during a critical period, while 2 days of light exposure eliminated iLTD in both layers. Furthermore, the GABAergic synapses in layers 2/3 and 5 did not normally mature in the type 1 cannabinoid receptor knock-out mice, whereas those in layer 4 did not require endocannabinoid receptor for maturation. These results suggest that visually induced endocannabinoid-dependent iLTD mediates the maturation of GABAergic release in extragranular layer rather than in granular layer of mouse visual cortex.