Theta Frequency Electromagnetic Stimulation Enhances Functional Recovery After Stroke.

Extremely low-frequency, low-intensity electromagnetic field (ELF-EMF) therapy is a non-invasive brain stimulation method that can modulate neuroprotection and neuroplasticity. ELF-EMF was recently shown to enhance recovery in human stroke in a small pilot clinical trial (NCT04039178). ELF-EMFs encompass a wide range of frequencies, typically ranging from 1 to 100 Hz, and their effects can vary depending on the specific frequency employed. However, whether and to what extent the effectiveness of ELF-EMFs depends on the frequency remains unclear. In the present study, we aimed to assess the efficacy of different frequency-intensity protocols of ELF-EMF in promoting functional recovery in a mouse cortical stroke model with treatment initiated 4 days after the stroke, employing a series of motor behavior tests. Our findings demonstrate that a theta-frequency ELF-EMF (5 Hz) effectively enhances functional recovery in a reach-to-grasp task, whereas neither gamma-frequency (40 Hz) nor combination frequency (5-16-40 Hz) ELF-EMFs induce a significant effect. Importantly, our histological analysis reveals that none of the ELF-EMF protocols employed in our study affect infarct volume, inflammatory, or glial activation, suggesting that the observed beneficial effects may be mediated through non-neuroprotective mechanisms. Our data indicate that ELF-EMFs have an influence on functional recovery after stroke, and this effect is contingent upon the specific frequency used. These findings underscore the critical importance of optimizing the protocol parameters to maximize the beneficial effects of ELF-EMF. Further research is warranted to elucidate the underlying mechanisms and refine the protocol parameters for optimal therapeutic outcomes in stroke rehabilitation.

A novel theoretical framework for simultaneous measurement of excitatory and inhibitory conductances.

The firing of neurons throughout the brain is determined by the precise relations between excitatory and inhibitory inputs, and disruption of their balance underlies many psychiatric diseases. Whether or not these inputs covary over time or between repeated stimuli remains unclear due to the lack of experimental methods for measuring both inputs simultaneously. We developed a new analytical framework for instantaneous and simultaneous measurements of both the excitatory and inhibitory neuronal inputs during a single trial under current clamp recording. This can be achieved by injecting a current composed of two high frequency sinusoidal components followed by analytical extraction of the conductances. We demonstrate the ability of this method to measure both inputs in a single trial under realistic recording constraints and from morphologically realistic CA1 pyramidal model cells. Future experimental implementation of our new method will facilitate the understanding of fundamental questions about the health and disease of the nervous system.

Author Correction: Dynamics of social representation in the mouse prefrontal cortex.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dynamics of social representation in the mouse prefrontal cortex.

The prefrontal cortex (PFC) plays an important role in regulating social functions in mammals, and its dysfunction has been linked to social deficits in neurodevelopmental disorders. Yet little is known of how the PFC encodes social information and how social representations may be altered in such disorders. Here, we show that neurons in the medial PFC of freely behaving male mice preferentially respond to socially relevant olfactory cues. Population activity patterns in this region differed between social and nonsocial stimuli and underwent experience-dependent refinement. In mice lacking the autism-associated gene Cntnap2, both the categorization of sensory stimuli and the refinement of social representations were impaired. Noise levels in spontaneous population activity were higher in Cntnap2 knockouts and correlated with the degree to which social representations were disrupted. Our findings elucidate the encoding of social sensory cues in the medial PFC and provide a link between altered prefrontal dynamics and autism-associated social dysfunction.

Odor Concentration Change Coding in the Olfactory Bulb.

Dynamical changes in the environment strongly impact our perception. Likewise, sensory systems preferentially represent stimulus changes, enhancing temporal contrast. In olfaction, odor concentration changes across consecutive inhalations (ΔCt ) can guide odor source localization, yet the neural representation of ΔCt has not been studied in vertebrates. We have found that, in the mouse olfactory bulb, a subset of mitral/tufted (M/T) cells represents ΔCt , enhancing the contrast between different concentrations. These concentration change responses are direction selective: they respond either to increments or decrements of concentration, reminiscent of ON and OFF selectivity in the retina. This contrast enhancement scales with the magnitude, but not the duration of the concentration step. Further, ΔCt can be read out from the total spike count per sniff, unlike odor identity and intensity, which are represented by fast temporal spike patterns. Our results demonstrate that a subset of M/T cells represents ΔCt , providing a signal that may instruct navigational decisions in downstream olfactory circuits.

Volume Conduction Coupling of Whisker-Evoked Cortical LFP in the Mouse Olfactory Bulb.

Local field potentials (LFPs) are an important measure of brain activity and have been used to address various mechanistic and behavioral questions. We revealed a prominent whisker-evoked LFP signal in the olfactory bulb and investigated its physiology. This signal, dependent on barrel cortex activation and highly correlated with its local activity, represented a pure volume conduction signal that was sourced back to the activity in the ventro-lateral orbitofrontal cortex, located a few millimeters away. Thus, we suggest that special care should be taken when acquiring and interpreting LFP data.

Extracellular ATP Selectively Upregulates Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 and Ecto-5'-Nucleotidase by Rat Cortical Astrocytes In Vitro.

Extracellular ATP (eATP) acts as a danger-associated molecular pattern which induces reactive response of astrocytes after brain insult, including morphological remodeling of astrocytes, proliferation, chemotaxis, and release of proinflammatory cytokines. The responses induced by eATP are under control of ecto-nucleotidases, which catalyze sequential hydrolysis of ATP to adenosine. In the mammalian brain, ecto-nucleotidases comprise three enzyme families: ecto-nucleoside triphosphate diphosphohydrolases 1-3 (NTPDase1-3), ecto-nucleotide pyrophosphatase/phospodiesterases 1-3 (NPP1-3), and ecto-5'-nucleotidase (eN), which crucially determine ATP/adenosine ratio in the pericellular milieu. Altered expression of ecto-nucleotidases has been demonstrated in several experimental models of human brain dysfunctions. In the present study, we have explored the pattern of NTPDase1-3, NPP1-3, and eN expression by cultured cortical astrocytes challenged with 1 mmol/L ATP (eATP). At the transcriptional level, eATP upregulated expression of NTPDase1, NTPDase2, NPP2, and eN, while, at translational and functional levels, these were paralleled only by the induction of NTPDase2 and eN. Additionally, eATP altered membrane topology of eN, from clusters localized in membrane domains to continuous distribution along the cell membrane. Our results suggest that eATP, by upregulating NTPDase2 and eN and altering the enzyme membrane topology, affects local kinetics of ATP metabolism and signal transduction that may have important roles in the process related to inflammation and reactive gliosis.

Repetitive hyperbaric oxygenation attenuates reactive astrogliosis and suppresses expression of inflammatory mediators in the rat model of brain injury.

The exact mechanisms by which treatment with hyperbaric oxygen (HBOT) exerts its beneficial effects on recovery after brain injury are still unrevealed. Therefore, in this study we investigated the influence of repetitive HBOT on the reactive astrogliosis and expression of mediators of inflammation after cortical stab injury (CSI). CSI was performed on male Wistar rats, divided into control, sham, and lesioned groups with appropriate HBO. The HBOT protocol was as follows: 10 minutes of slow compression, 2.5 atmospheres absolute (ATA) for 60 minutes, and 10 minutes of slow decompression, once a day for 10 consecutive days. Data obtained using real-time polymerase chain reaction, Western blot, and immunohistochemical and immunofluorescence analyses revealed that repetitive HBOT applied after the CSI attenuates reactive astrogliosis and glial scarring, and reduces expression of GFAP (glial fibrillary acidic protein), vimentin, and ICAM-1 (intercellular adhesion molecule-1) both at gene and tissue levels. In addition, HBOT prevents expression of CD40 and its ligand CD40L on microglia, neutrophils, cortical neurons, and reactive astrocytes. Accordingly, repetitive HBOT, by prevention of glial scarring and limiting of expression of inflammatory mediators, supports formation of more permissive environment for repair and regeneration.

Effect of stab injury in the rat cerebral cortex on temporal pattern of expression of neuronal cytoskeletal proteins: an immunohistochemical study.

Compelling evidence now points to the critical role of the cytoskeleton in neurodegeneration. In the present study, using an immunohistochemical approach, we have shown that cortical stab injury (CSI) in adult Wistar rats significantly affects temporal pattern of expression of neurofilament proteins (NFs), a major cytoskeleton components of neurons, and microtubule-associated proteins (MAP2). At 3 days post-injury (dpi) most of the NFs immunoreactivity was found in pyknotic neurons and in fragmentized axonal processes in the perilesioned cortex. These cytoskeletal alterations became more pronounced by 10dpi. At the subcellular level CSI also showed significant impact on NFs and MAP-2 expression. Thus, at 3dpi most of the dendrites disappeared, while large neuronal somata appeared like open circles pointing to membrane disintegration. Conversely, at 10dpi neuronal perikarya and a few new apical dendrites were strongly labeled. Since aberrant NF phosphorylation is a pathological hallmark of many human neurodegenerative disorders, as well as is found after stressor stimuli, the present results shed light into the expression of neurofilaments after the stab brain injury.

Ribavirin shows immunomodulatory effects on activated microglia.

Abstract Ribavirin (RBV) is synthetic purine nucleoside analogue, licensed as anti-viral drug that displays immunomodulatory actions on various immune cells. Our previous ex vivo studies have demonstrated immunosuppressive effects of RBV on reactive T-lymphocytes in experimental autoimmune encephalomyelitis. Here, we examined the effects of RBV on inflammatory response of microglia. RBV potency to down-regulate microglia inflammatory response was assessed by measuring microglia cell body size, and the production of nitric oxide (NO) and pro- and anti-inflammatory cytokines. RBV exerted cytotoxic effects on LPS-stimulated microglia, leaving non-stimulated microglia unaffected. The exposure of activated microglia to RBV led to: decrease in the level of NO as a result of decreased cell number, lower average cell surface, the reduction of membrane ruffling, the suppression of interleukin-6 release and promoted interleukin-10 production. On the other hand, RBV promoted LPS-induced interleukin-1 beta release. Our results imply that RBV is a complex immunomodulator showing both anti- and pro-inflammatory effects on activated microglia.

Fluctuating vs. continuous exposure to H2O2: the effects on mitochondrial membrane potential, intracellular calcium, and NF-κB in astroglia.

The effects of H2O2 are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H2O2 concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H2O2, using three regimes of exposure - continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity - respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H2O2 induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H2O2 provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H2O2 induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.

The effect of ribavirin on reactive astrogliosis in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model of CNS inflammatory and demyelinating disease multiple sclerosis. Microglia and astrocytes represent two related cell types involved in the brain pathology in EAE. Accumulations of hypertrophic reactive astrocytes, intensely stained with glial fibrillary acidic protein (GFAP), which also expressed vimentin, are prominent features of EAE lesions. Recent studies from our laboratory reported that ribavirin attenuated the disease process in EAE by reducing clinical and histological manifestations. EAE was induced in genetically susceptible Dark Agouti rats with syngeneic spinal cord homogenate in complete Freund's adjuvant. Real time PCR and immunohistochemistry were used for determination of GFAP and vimentin gene and tissue expression. We have observed the increased gene and tissue expression of GFAP and vimentin in EAE rats. Ribavirin treatment significantly decreased the number of reactive astrocytes at the peak of disease. At the end of the disease, we have observed reactive GFAP(+) and vimentin(+) astrocytes in both immunized and ribavirin-treated groups, accompanied by increased level of GFAP mRNA. The present study indicates that ribavirin may have the ability to attenuate astrocyte proliferation and glial scaring at the peak of the disease and modulate the astroglial response to EAE during the time-course of the disease.

Hyperbaric oxygenation improves locomotor ability by enhancing neuroplastic responses after cortical ablation in rats.

OBJECTIVE: To investigate whether hyperbaric oxygenation (HBO) can improve the recovery of motor functions in rats after suction ablation of the right sensorimotor cortex. METHODS: The experimental paradigm implies the following groups: Control animals (C), Control + HBO (CHBO), Sham controls (S), Sham control + HBO (SHBO), Lesion group (L), right sensorimotor cortex was removed by suction, Lesion + HBO (LHBO). Hyperbaric protocol: pressure applied 2.5 atmospheres absolute, for 60 minutes, once a day for 10 days. A beam walking test and grip strength meter were used to evaluate the recovery of motor functions. Expression profiles of growth-associated protein 43 (GAP43) and synaptophysin (SYP) were detected using immunohistochemistry. RESULTS: The LHBO group achieved statistically superior scores in the beam walking test compared to the L group. Additionally, the recovery of muscle strength of the affected hindpaw was significantly enhanced after HBO treatment. Hyperbaric oxygenation induced over-expression of GAP43 and SYP in the neurons surrounding the lesion site. CONCLUSIONS: Data presented suggest that hyperbaric oxygen therapy can intensify neuroplastic responses by promoting axonal sprouting and synapse remodelling, which contributes to the recovery of locomotor performances in rats. This provides the perspective for implementation of HBO in clinical strategies for treating traumatic brain injuries.

Hyperbaric oxygenation alters temporal expression pattern of superoxide dismutase 2 after cortical stab injury in rats.

AIM: To evaluate the effect of hyperbaric oxygen therapy (HBOT) on superoxide dismutase 2 (SOD2) expression pattern after the cortical stab injury (CSI). METHODS: CSI was performed on 88 male Wistar rats, divided into control, sham, lesioned, and HBO groups. HBOT protocol was the following: pressure applied was 2.5 absolute atmospheres, for 60 minutes, once a day for consecutive 3 or 10 days. The pattern of SOD2 expression and cellular localization was analyzed using real-time polymerase chain reaction, Western blot, and double-label fluorescence immunohistochemistry. Neurons undergoing degeneration were visualized with Fluoro-Jade®B. RESULTS: CSI induced significant transient increase in SOD2 protein levels at day 3 post injury, which was followed by a reduction toward control levels at post-injury day 10. At the same time points, mRNA levels for SOD2 in the injured cortex were down-regulated. Exposure to HBO for 3 days considerably down-regulated SOD2 protein levels in the injured cortex, while after 10 days of HBOT an up-regulation of SOD2 was observed. HBOT significantly increased mRNA levels for SOD2 at both time points compared to the corresponding L group, but they were still lower than in controls. Double immunofluorescence staining revealed that 3 days after CSI, up-regulation of SOD2 was mostly due to an increased expression in reactive astrocytes surrounding the lesion site. HBOT attenuated SOD2 expression both in neuronal and astroglial cells. Fluoro-Jade®B labeling showed that HBOT significantly decreased the number of degenerating neurons in the injured cortex. CONCLUSION: HBOT alters SOD2 protein and mRNA levels after brain injury in a time-dependent manner.

Dynamic changes in the expression pattern of ecto-5'-nucleotidase in the rat model of cortical stab injury.

Traumatic injury induces massive release of ATP in the extracellular space, where it influences numerous aspects of neuronal, astrocytic, and microglial responses to injury by activating P2X and P2Y receptors. The extracellular ATP actions are controlled by the ectonucleotidase enzyme pathway, which hydrolyses ATP to adenosine at all neuronal and nonneuronal cell types. Adenosine activates its P1 receptors, which have important neuroprotective roles. The rate-limiting enzyme in the ectonucleotidase pathway is ecto-5'-nucleotidase (e-5NT), which catalyzes the final step of dephosphorylation of AMP to adenosine. The aim of the present study was to characterize the expression pattern and cellular distribution of e-5NT in the perilesioned cortex at 4 hr and 1, 2, 7, and 15 days after unilateral cortical stab injury (CSI). Immunoblot and immunohistochemical studies showed that overall e-5NT expression was lower 4 hr and 1 day postinjury and then gradually increased above the control levels. Double-immunofluorescence studies further showed in control tissue the presence of the enzyme in the membranes surrounding neuronal somata and apical dendrites and less frequently in astrocytes. CSI caused a rapid (after 4 hr) and irreversible loss of the enzyme from neurons, accounting for a decrease in the overall enzyme expression. This was accompanied with a gradual increase in e-5NT-positive astrocytes, accounting for up-regulation of the enzyme levels in the injured area. Thus, CSI induced dynamic changes in the expression pattern of e-5NT that modify the ATP/adenosine ratio and the extent of P1 and P2 receptors activation and, therefore, outcome of the pathological processes after CSI.