Axon regeneration in the central nervous system is severely hampered, limiting functional recovery. This is in part because of endogenous axon regeneration inhibitors that accumulate at the injury site. Therapeutic targeting of these inhibitors and their receptors may facilitate axon outgrowth and enhance recovery. A rat model of spinal cord contusion injury was used to test the effects of two bacterial enzyme therapies that target independent axon regeneration inhibitors, sialidase (Vibrio cholerae) and chondroitinase ABC (ChABC, Proteus vulgaris). The two enzymes, individually and in combination, were infused for 2 weeks via implanted osmotic pumps to the site of a moderate thoracic spinal cord contusion injury. Sialidase was completely stable, whereas ChABC retained>30% of its activity in vivo over the 2 week infusion period. Immunohistochemistry revealed that infused sialidase acted robustly throughout the spinal cord gray and white matter, whereas ChABC activity was more intense superficially. Sialidase treatment alone resulted in improved behavioral and anatomical outcomes. Rats treated exclusively with sialidase showed significantly increased hindlimb motor function, evidenced by higher Basso Beattie and Bresnahan (BBB) and BBB subscores, and fewer stepping errors on a horizontal ladder. Sialidase-treated rats also had increased serotonergic axons caudal to the injury. ChABC treatment, in contrast, did not enhance functional recovery or alter axon numbers after moderate spinal cord contusion injury, and dampened the response of sialidase in the dual enzyme treatment group. We conclude that sialidase infusion enhanced recovery from spinal cord contusion injury, and that combining sialidase with ChABC failed to improve outcomes.

Gangliosides - sialic acid-bearing glycolipids - are major cell surface determinants on neurons and axons. The same four closely related structures, GM1, GD1a, GD1b and GT1b, comprise the majority of total brain gangliosides in mammals and birds. Gangliosides regulate the activities of proteins in the membranes in which they reside, and also act as cell-cell recognition receptors. Understanding the functions of major brain gangliosides requires knowledge of their tissue distribution, which has been accomplished in the past using biochemical and immunohistochemical methods. Armed with new knowledge about the stability and accessibility of gangliosides in tissues and new IgG-class specific monoclonal antibodies, we investigated the detailed tissue distribution of gangliosides in the adult mouse brain. Gangliosides GD1b and GT1b are widely expressed in gray and white matter. In contrast, GM1 is predominately found in white matter and GD1a is specifically expressed in certain brain nuclei/tracts. These findings are considered in relationship to the hypothesis that gangliosides GD1a and GT1b act as receptors for an important axon-myelin recognition protein, myelin-associated glycoprotein (MAG). Mediating axon-myelin interactions is but one potential function of the major brain gangliosides, and more detailed knowledge of their distribution may help direct future functional studies.

Adult zebrafish (Danio rerio) have a remarkable ability to restore function after an injury to the brain or spinal cord. The molecular and cellular mechanisms underlying this phenomenon are not fully understood. To enable investigation of these mechanisms we have developed an in vitro model system from the adult zebrafish brainstem, which can be maintained under serum-containing and serum-free conditions. While cultures are predominantly neuronal, they also contain glia and stem progenitor cells. Various stages of cellular differentiation are observed among both neuronal and non-neuronal populations. Quantitative morphological results revealed typical cellular growth over a two-week period. We argue that our novel brainstem culture model offers a powerful tool for the studies of axonal growth, neurogenesis, and regeneration in the adult zebrafish central nervous system.

Regenerated cerebrospinal axons are considered to be involved in the spontaneous recovery of swimming ability following a spinal cord injury in adult zebrafish. We employed behavioral analysis, neuronal tracing, and immunocytochemistry to determine the exact temporal relationship between swimming ability and regenerated cerebrospinal axon number in adult zebrafish with a complete spinal cord transection. Between two and eight weeks post-lesion, swimming gradually improved to 44% of sham-injured zebrafish. Neurons within the reticular formation, magnocellular octaval nucleus, and nucleus of the medial longitudinal fascicle grew their axon across and at least four millimeters beyond the lesion. The largest increases in swimming ability and number of regenerated cerebrospinal axons were observed between two and four weeks post-lesion. Regression analyses revealed a significant correlation between swimming ability and the number of regenerated axons. Our results indicate the involvement of cerebrospinal axons in swimming recovery after spinal cord injury in adult zebrafish.

Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.

Freeze-dried poly(D,L-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with brain-derived neurotrophic factor and neurotrophin-3 activity, and to express green fluorescent protein (GFP) were implanted in the completely transected adult rat thoracic spinal cord. Control rats were similarly injured and then implanted with scaffolds containing the fibrin solution with SCs lentivirally transduced to produce express GFP only or with the fibrin solution only. Transgene production and biological activity in vitro, SC survival within the scaffold in vitro and in vivo, scaffold integration, axonal regeneration and myelination, and hind limb motor function were analyzed at 1, 2, and 6 weeks after implantation. In vitro, lentivirally transduced SCs produced 87.5 ng/24 h/10(6) cells of D15A as measured by neurotrophin-3 activity in ELISA. The secreted D15A was biologically active as evidenced by its promotion of neurite outgrowth of dorsal root ganglion neurons in culture. In vitro, SCs expressing GFP were present in the scaffolds for up to 6 h, the end of a typical surgery session. Implantation of SC-seeded scaffolds caused modest loss of spinal nervous tissue. Reactive astrocytes and chondroitin sulfate glycosaminoglycans were present in spinal tissue adjacent to the scaffold. Vascularization of the scaffold was ongoing at 1 week post-implantation. There were no apparent differences in scaffold integration and blood vessel formation between groups. A decreasing number of implanted (GFP-positive) SCs were found within the scaffold during the first 3 days after implantation. Apoptosis was identified as one of the mechanisms of cell death. At 1 week and later time points after implantation, few of the implanted SCs were present in the scaffold. Neurofilament-positive axons were found in the scaffold. At 6 weeks post-grafting, myelinated axons were observed within and at the external surface of the scaffold. Axons did not grow from the scaffold into the caudal cord. All groups demonstrated a similar improvement of hind limb motor function. Our findings demonstrated that few seeded SCs survived in vivo, which could account for the modest axonal regeneration response into and across the scaffold. For the development of SC-seeded macroporous scaffolds that effectively promote axonal regeneration in the injured spinal cord, the survival and/or total number of SCs in the scaffold needs to be improved.

It was investigated whether the addition of basic fibroblast growth factor (FGF-2) enhances the efficacy of a Schwann cell (SC) bridge to repair the transected spinal cord by assessing tissue sparing and neuronal survival near the graft-cord interfaces, axonal regeneration and myelination in the graft, and behavioral recovery up to 12 weeks post-grafting. Experimental animals received a bridge of SCs within fibrin containing 1 microg of FGF-2; control animals received a SC implant without FGF-2. Sparing of tissue in a 2.5-mm-long segment near the graft-cord borders was 69% in the rostral and 52% in the caudal cord at 6 weeks post-grafting, not significantly different from the control group. With FGF-2, survival of NeuN-positive cells was increased in the rostral cord: 24.4%, 20.4%, and 17.2% of the number of positive cells in the uninjured cord compared to 13.5%, 9.1%, and 8.9% in controls at 3, 6, and 12 weeks post-grafting, respectively. Similarly, in the caudal cord, survival of NeuN-positive cells was increased with FGF-2: 19.3%, 16.8%, and 14.5% compared to 10.8%, 5.6%, and 6.1% in controls. The staining intensity of glial fibrillary acidic protein was significantly higher at the interfaces of both cord stumps at 3 weeks with SC/FGF-2 grafts; chondroitin sulfate proteoglycan (CS-56) staining was more intense in the rostral cord but only at 6 weeks. Blood vessels in the FGF-2 grafts were larger and less regular in shape than those in control grafts. Axonal growth into the bridge was not improved by the addition of FGF-2. Retrogradely traced neurons were not found rostral to the implant, indicating that axons had not grown a few mm into the caudal spinal tissue. Recovery of hind limb function was similar in both groups. Despite the neuroprotective effects of FGF-2, improved effects on axonal regeneration and functional recovery were not observed.

We previously showed that Nuclear Factor kappaB (NF-kappaB) inactivation in astrocytes leads to improved functional recovery following spinal cord injury (SCI). This correlated with reduced expression of pro-inflammatory mediators and chondroitin sulfate proteoglycans, and increased white matter preservation. Hence we hypothesized that inactivation of astrocytic NF-kappaB would create a more permissive environment for axonal sprouting and regeneration. We induced both contusive and complete transection SCI in GFAP-Inhibitor of kappaB-dominant negative (GFAP-IkappaBalpha-dn) and wild-type (WT) mice and performed retrograde [fluorogold (FG)] and anterograde [biotinylated dextran amine (BDA)] tracing 8 weeks after injury. Following contusive SCI, more FG-labeled cells were found in motor cortex, reticular formation, and raphe nuclei of transgenic mice. Spared and sprouting BDA-positive corticospinal axons were found caudal to the lesion in GFAP-IkappaBalpha-dn mice. Higher numbers of FG-labeled neurons were detected immediately rostral to the lesion in GFAP-IkappaBalpha-dn mice, accompanied by increased expression of synaptic and axonal growth-associated molecules. After transection, however, no FG-labeled neurons or BDA-filled axons were found rostral and caudal to the lesion, respectively, in either genotype. These data demonstrated that inhibiting astroglial NF-kappaB resulted in a growth-supporting terrain promoting sparing and sprouting, rather than regeneration, of supraspinal and propriospinal circuitries essential for locomotion, hence contributing to the improved functional recovery observed after SCI in GFAP-IkappaBalpha-dn mice.

Crossed facilitatory interactions in the corticospinal pathway are impaired in humans with chronic incomplete spinal cord injury (SCI). The extent to which crossed facilitation is affected in muscles above and below the injury remains unknown. To address this question we tested 51 patients with neurological injuries between C2-T12 and 17 age-matched healthy controls. Using transcranial magnetic stimulation we elicited motor evoked potentials (MEPs) in the resting first dorsal interosseous, biceps brachii, and tibialis anterior muscles when the contralateral side remained at rest or performed 70% of maximal voluntary contraction (MVC) into index finger abduction, elbow flexion, and ankle dorsiflexion, respectively. By testing MEPs in muscles with motoneurons located at different spinal cord segments we were able to relate the neurological level of injury to be above, at, or below the location of the motoneurons of the muscle tested. We demonstrate that in patients the size of MEPs was increased to a similar extent as in controls in muscles above the injury during 70% of MVC compared to rest. MEPs remained unchanged in muscles at and within 5 segments below the injury during 70% of MVC compared to rest. However, in muscles beyond 5 segments below the injury the size of MEPs increased similar to controls and was aberrantly high, 2-fold above controls, in muscles distant (>15 segments) from the injury. These aberrantly large MEPs were accompanied by larger F-wave amplitudes compared to controls. Thus, our findings support the view that corticospinal degeneration does not spread rostral to the lesion, and highlights the potential of caudal regions distant from an injury to facilitate residual corticospinal output after SCI.

Axons fail to regenerate in the injured spinal cord, limiting motor and autonomic recovery and contributing to long-term morbidity. Endogenous inhibitors, including those on residual myelin, contribute to regeneration failure. One inhibitor, myelin-associated glycoprotein (MAG), binds to sialoglycans and other receptors on axons. MAG inhibition of axon outgrowth in some neurons is reversed by treatment with sialidase, an enzyme that hydrolyzes sialic acids and eliminates MAG-sialoglycan binding. We delivered recombinant sialidase intrathecally to rats following a spinal cord contusive injury. Sialidase (or saline solution) was infused to the injury site continuously for 2 wk and then motor behavior, autonomic physiology, and anatomic outcomes were determined 3 wk later. Sialidase treatment significantly enhanced hindlimb motor function, improved bulbospinally mediated autonomic reflexes, and increased axon sprouting. These findings validate sialoglycans as therapeutic targets and sialidase as a candidate therapy for spinal cord injury.

Methylprednisolone (MP) and interleukin-10 (IL-10) are tissue protective acutely after spinal cord injury (SCI); their combination offers additive protection (Takami et al., 2002a). Our study examined if acute administration of MP (30 mg/kg i.v. at 5 min, and 2 and 4 h after injury) and IL-10 (30 mg/kg i.p. at 30 min after injury) increases the efficacy of Schwann cell (SC) or SC plus olfactory ensheathing glia (SC/OEG) grafts transplanted into rat thoracic cord 1 week after contusive injury. Efficacy was determined by histology, anterograde and retrograde tracing, immunohistochemistry for gliosis and specific nerve fibers, and several behavioral tests. Administration of MP/IL-10 or SC or SC/OEG transplantation significantly increased the total volume of a 9-mm segment of cord encompassing the injury site at 12 weeks. The combination of either SC or SC/OEG transplantation with MP/IL-10 most significantly reduced cavitation. The individual treatments all significantly increased the volume of normal-appearing tissue compared to injury-only controls; however, significant decreases in the volume of normal-appearing tissue were seen when MP/IL-10 and cell grafts were combined compared to MP/IL-10 alone. SC/OEG grafts were effective in promoting serotonergic fiber growth into the graft and led to more reticulospinal fibers caudal to the graft; combination with MP/IL-10 did not further increase fiber number. Only the combination of MP/IL-10 with SC/OEG transplants significantly improved gross locomotor performance (BBB scores) over injury-only controls. MP/IL-10 given prior to SC-only transplants, however, worsened behavioral outcome. Because beneficial effects of MP/IL-10 were not always additive when combined with cell transplantation, we need to understand (1) how tissue protective agents may transform the milieu of the injured spinal cord to the benefit or detriment of later transplanted cells and (2) whether neuroprotectants need to be re-administered at the time of cell grafting or less invasive transplantation techniques employed to reduce damage to tissue spared by an earlier protection strategy.

An injury to the spinal cord causes long-lasting loss of nervous tissue because endogenous nervous tissue repair and regeneration at the site of injury is limited. We engineered an injectable nanofiber-hydrogel composite (NHC) with interfacial bonding to provide mechanical strength and porosity and examined its effect on repair and neural tissue regeneration in an adult rat model of spinal cord contusion. At 28 days after treatment with NHC, the width of the contused spinal cord segment was 2-fold larger than in controls. With NHC treatment, tissue in the injury had a 2-fold higher M2/M1 macrophage ratio, 5-fold higher blood vessel density, 2.6-fold higher immature neuron presence, 2.4-fold higher axon density, and a similar glial scar presence compared with controls. Spared nervous tissue volume in the contused segment and hind limb function was similar between groups. Our findings indicated that NHC provided mechanical support to the contused spinal cord and supported pro-regenerative macrophage polarization, angiogenesis, axon growth, and neurogenesis in the injured tissue without any exogenous factors or cells. These results motivate further optimization of the NHC and delivery protocol to fully translate the potential of the unique properties of the NHC for treating spinal cord injury.

A bone marrow-derived mesenchymal stromal cell (MSC) transplant and a bioengineered nanofiber-hydrogel composite (NHC) have been shown to stimulate nervous tissue repair in the contused spinal cord in rodent models. Here, these two modalities were combined to assess their repair effects in the contused spinal cord in adult rats. Cohorts of contused rats were treated with MSC in NHC (MSC-NHC), MSC in phosphate-buffered saline (MSC-PBS), NHC, or PBS injected into the contusion site at 3 days post-injury. One week after injury, there were significantly fewer CD68+ cells in the contusion with MSC-NHC and NHC, but not MSC-PBS. The reduction in CD86+ cells in the injury site with MSC-NHC was mainly attributed to NHC. One and eight weeks after injury, we found a greater CD206+/CD86+ cell ratio with MSC-NHC or NHC, but not MSC-PBS, indicating a shift from a pro-inflammatory towards an anti-inflammatory milieu in the injury site. Eight weeks after injury, the injury size was significantly reduced with MSC-NHC, NHC, and MSC-PBS. At this time, astrocyte, and axon presence in the injury site was greater with MSC-NHC compared with MSC-PBS. We did not find a significant effect of NHC on MSC transplant survival, and hind limb function was similar across all groups. However, we did find fewer macrophages at 1 week post-injury, more macrophages polarized towards a pro-regenerative phenotype at 1 and 8 weeks after injury, and reduced injury volume, more astrocytes, and more axons at 8 weeks after injury in rats with MSC-NHC and NHC alone compared with MSC-PBS; these findings were especially significant between rats with MSC-NHC and MSC-PBS. The data support further study in the use of an NHC-MSC combination transplant in the contused spinal cord.

Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5' and 3' cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 10(11) genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.

Cellular therapies represent a novel treatment approach for spinal cord injury (SCI), with many different cellular substrates showing promise in preclinical animal models of SCI. Considerable interest therefore exists to translate such cellular interventions into human clinical trials. Balanced against the urgency for clinical translation is the desire to establish the robustness of a cellular therapy's efficacy in preclinical studies, thereby optimizing its chances of succeeding in human trials. Uncertainty exists, however, on the extent to which a therapy needs to demonstrate efficacy in the preclinical setting in order to justify the initiation of a lengthy, expensive, and potentially risky clinical trial. The purpose of this initiative was to seek perspectives on the level of evidence required in experimental studies of cellular therapies before proceeding with clinical trials of SCI. We conducted a survey of 27 SCI researchers actively involved in either preclinical and/or clinical research of cellular interventions for SCI, and then held a focus group meeting to facilitate more in-depth discussion around a number of translational issues. These included: the use of animal models, the use of injury models and mechanisms, the window for demonstrating efficacy, independent replication, defining "relevant, meaningful efficacy" in preclinical studies, and the expectation of therapeutic benefits for cellular interventions. Here we present the key findings from both the survey and focus group meeting in order to summarize and underscore the areas of consensus and disagreement amongst the sampled researchers. It is anticipated that the knowledge generated from this initiative will help to incite future scientific discussions and expert guidelines towards translation of a cell therapy for persons with SCI.

We sought to directly compare growth and myelination of local and supraspinal axons by implanting into the injured spinal cord Schwann cells (SCs) transduced ex vivo with adenoviral (AdV) or lentiviral (LV) vectors encoding a bifunctional neurotrophin molecule (D15A). D15A mimics actions of both neurotrophin-3 and brain-derived neurotrophic factor. Transduced SCs were injected into the injury center 1 week after a moderate thoracic (T8) adult rat spinal cord contusion. D15A expression and bioactivity in vitro; D15A levels in vivo; and graft volume, SC number, implant axon number and cortico-, reticulo-, raphe-, coerulo-spinal and sensory axon growth were determined for both types of vectors employed to transduce SCs. ELISAs revealed that D15A-secreting SC implants contained significantly higher levels of neurotrophin than non-transduced SC and AdV/GFP and LV/GFP SC controls early after implantation. At 6 weeks post-implantation, D15A-secreting SC grafts exhibited 5-fold increases in graft volume, SC number and myelinated axon counts and a 3-fold increase in myelinated to unmyelinated (ensheathed) axon ratios. The total number of axons within grafts of LV/GFP/D15A SCs was estimated to be over 70,000. Also 5-HT, DbetaH, and CGRP axon length was increased up to 5-fold within D15A grafts. In sum, despite qualitative differences using the two vectors, increased neurotrophin secretion by the implanted D15A SCs led to the presence of a significantly increased number of axons in the contusion site. These results demonstrate the therapeutic potential for utilizing neurotrophin-transduced SCs to repair the injured spinal cord.

Gene therapy is being developed for the treatment of inherited diseases, whereby a therapeutic gene is continuously expressed in patients after delivery via viral vectors such as adeno-associated virus (AAV). Depending on the transgene, there could be a limited therapeutic window, and regulating timing and levels of transgene expression is advantageous. To control transgene transcription, the regulatory system GeneSwitch (GS) was evaluated in detail both in vitro and in vivo. The classical two-plasmid mifepristone (MFP)-inducible GS system was put into one plasmid or a single AAV5 vector. Our data demonstrate the inducibility of multiple transgenes and the importance of promoter and regulatory elements within the GS system. Mice injected with AAV5 containing the GS system transiently expressed mRNA and protein after MFP induction. The inducer MFP could be measured in plasma and liver tissue, and assessment of MFP and its metabolites showed rapid clearance from murine plasma. In a head-to-head comparison, our single vector outclassed the classical two-vector GS system. Finally, we show repeated inducibility of the transgene that also translated into a dynamic phenotypic change in mice. Taken together, this in-depth analysis of the GS system shows its applicability for regulated gene therapy.

Of the adeno-associated viruses (AAVs), AAV9 is known for its capability to cross the blood-brain barrier (BBB) and can, therefore, be used as a noninvasive method to target the central nervous system. Furthermore, the addition of the peptide PhP.B to AAV9 increases its transduction across the BBB by 40-fold. Another neurotropic serotype, AAV5, has been shown as a gene therapeutic delivery vehicle to ameliorate several neurodegenerative diseases in preclinical models, but its administration requires invasive surgery. In this study, AAV9-PhP.B and AAV5-PhP.B were designed and produced in an insect cell-based system. To AAV9, the PhP.B peptide TLAVPFK was added, whereas in AAV5-PhP.B (AQTLAVPFKAQAQ), with AQ-AQAQ sequences used to swap with the corresponding sequence of AAV5. The addition of PhP.B to AAV5 did not affect its capacity to cross the mouse BBB, while increased transduction of liver tissue was observed. Then, intravenous (IV) and intrastriatal (IStr) delivery of AAV9-PhP.B and AAV5 were compared. For AAV9-PhP.B, similar transduction and expression levels were achieved in the striatum and cortex, irrespective of the delivery method used. IStr administration of AAV5 resulted in significantly higher amounts of vector DNA and therapeutic miRNA in the target regions such as striatum and cortex when compared with an IV administration of AAV9-PhP.B. These results illustrate the challenge in developing a vector that can be delivered noninvasively while achieving a transduction level similar to that of direct administration of AAV5. Thus, for therapeutic miRNA delivery with high local expression requirements, intraparenchymal delivery of AAV5 is preferred, whereas a humanized AAV9-PhP.B may be useful when widespread brain (and peripheral) transduction is needed.

Mesenchymal stromal cells (MSC) are used for cell therapy for spinal cord injury (SCI) because of their ability to support tissue repair by paracrine signaling. Preclinical and clinical research testing MSC transplants for SCI have revealed limited success, which warrants the exploration of strategies to improve their therapeutic efficacy. MSC are sensitive to the microenvironment and their secretome can be altered in vitro by exposure to different culture media. Priming MSC with inflammatory stimuli increases the expression and secretion of reparative molecules. We studied the effect of macrophage-derived inflammation priming on MSC transplants and of primed MSC (pMSC) acute transplants (3 days) on spinal cord repair using an adult rat model of moderate-severe contusive SCI. We found a decrease in long-term survival of pMSC transplants compared with unprimed MSC transplants. With a pMSC transplant, we found significantly more anti-inflammatory macrophages in the contusion at 4 weeks post transplantation (wpt). Blood vessel presence and maturation in the contusion at 1 wpt was similar in rats that received pMSC or untreated MSC. Nervous tissue sparing and functional recovery were similar across groups. Our results indicate that macrophage-derived inflammation priming does not increase the overall therapeutic potential of an MSC transplant in the adult rat contused spinal cord.

Benzodiazepine (BZ) drugs treat seizures, anxiety, insomnia, and alcohol withdrawal by potentiating γ2 subunit containing GABA type A receptors (GABAARs). BZ clinical use is hampered by tolerance and withdrawal symptoms including heightened seizure susceptibility, panic, and sleep disturbances. Here, we investigated inhibitory GABAergic and excitatory glutamatergic plasticity in mice tolerant to benzodiazepine sedation. Repeated diazepam (DZP) treatment diminished sedative effects and decreased DZP potentiation of GABAAR synaptic currents without impacting overall synaptic inhibition. While DZP did not alter γ2-GABAAR subunit composition, there was a redistribution of extrasynaptic GABAARs to synapses, resulting in higher levels of synaptic BZ-insensitive α4-containing GABAARs and a concomitant reduction in tonic inhibition. Conversely, excitatory glutamatergic synaptic transmission was increased, and NMDAR subunits were upregulated at synaptic and total protein levels. Quantitative proteomics further revealed cortex neuroadaptations of key pro-excitatory mediators and synaptic plasticity pathways highlighted by Ca2+/calmodulin-dependent protein kinase II (CAMKII), MAPK, and PKC signaling. Thus, reduced inhibitory GABAergic tone and elevated glutamatergic neurotransmission contribute to disrupted excitation/inhibition balance and reduced BZ therapeutic power with benzodiazepine tolerance.