Transcranial static magnetic stimulation reduces seizures in a mouse model of Dravet syndrome.

Dravet syndrome is a rare form of severe genetic epilepsy characterized by recurrent and long-lasting seizures. It appears around the first year of life, with a quick evolution toward an increase in the frequency of the seizures, accompanied by a delay in motor and cognitive development, and does not respond well to antiepileptic medication. Most patients carry a mutation in the gene SCN1A encoding the α subunit of the voltage-gated sodium channel Nav1.1, resulting in hyperexcitability of neural circuits and seizure onset. In this work, we applied transcranial static magnetic stimulation (tSMS), a non-invasive, safe, easy-to-use and affordable neuromodulatory tool that reduces neural excitability in a mouse model of Dravet syndrome. We demonstrate that tSMS dramatically reduced the number of crises. Furthermore, crises recorded in the presence of the tSMS were shorter and less intense than in the sham condition. Since tSMS has demonstrated its efficacy at reducing cortical excitability in humans without showing unwanted side effects, in an attempt to anticipate a possible use of tSMS for Dravet Syndrome patients, we performed a numerical simulation in which the magnetic field generated by the magnet was modeled to estimate the magnetic field intensity reached in the cerebral cortex, which could help to design stimulation strategies in these patients. Our results provide a proof of concept for nonpharmacological treatment of Dravet syndrome, which opens the door to the design of new protocols for treatment.

Cross-modal reaction of auditory and visual cortices after long-term bilateral hearing deprivation in the rat.

Visual cortex (VC) over-activation analysed by evoked responses has been demonstrated in congenital deafness and after long-term acquired hearing loss in humans. However, permanent hearing deprivation has not yet been explored in animal models. Thus, the present study aimed to examine functional and molecular changes underlying the visual and auditory cross-modal reaction. For such purpose, we analysed cortical visual evoked potentials (VEPs) and the gene expression (RT-qPCR) of a set of markers for neuronal activation (c-Fos) and activity-dependent homeostatic compensation (Arc/Arg3.1). To determine the state of excitation and inhibition, we performed RT-qPCR and quantitative immunocytochemistry for excitatory (receptor subunits GluA2/3) and inhibitory (GABAA-α1, GABAB-R2, GAD65/67 and parvalbumin-PV) markers. VC over-activation was demonstrated by a significant increase in VEPs wave N1 and by up-regulation of the activity-dependent early genes c-Fos and Arc/Arg3.1 (thus confirming, by RT-qPCR, our previously published immunocytochemical results). GluA2 gene and protein expression were significantly increased in the auditory cortex (AC), particularly in layers 2/3 pyramidal neurons, but inhibitory markers (GAD65/67 and PV-GABA interneurons) were also significantly upregulated in the AC, indicating a concurrent increase in inhibition. Therefore, after permanent hearing loss in the rat, the VC is not only over-activated but also potentially balanced by homeostatic regulation, while excitatory and inhibitory markers remain imbalanced in the AC, most likely resulting from changes in horizontal intermodal regulation.

Sight and insight--on the physiological role of nitric oxide in the visual system.

Research in the fields of cellular communication and signal transduction in the brain has moved very rapidly in recent years. Nitric oxide (NO) is one of the latest discoveries in the arena of messenger molecules. Current evidence indicates that, in visual system, NO is produced in both postsynaptic and presynaptic structures and acts as a neurotransmitter, albeit of a rather unorthodox type. Under certain conditions it can switch roles to become either neuronal 'friend' or 'foe'. Nitric oxide is a gas that diffuses through all physiological barriers to act on neighbouring cells across an extensive volume on a specific time scale. It, therefore,has the opportunity to control the processing of vision from the lowest level of retinal transduction to the control of neuronal excitability in the visual cortex.

Specific roles of NMDA and AMPA receptors in direction-selective and spatial phase-selective responses in visual cortex.

Cells in the superficial layers of primary visual cortex (area 17) are distinguished by feedforward input from thalamic-recipient layers and by massive recurrent excitatory connections between neighboring cells. The connections use glutamate as transmitter, and the postsynaptic cells contain both NMDA and AMPA receptors. The possible role of these receptor types in generating emergent responses of neurons in the superficial cortical layers is unknown. Here, we show that NMDA and AMPA receptors are both involved in the generation of direction-selective responses in layer 2/3 cells of area 17 in cats. NMDA receptors contribute prominently to responses in the preferred direction, and their contribution to responses in the nonpreferred direction is reduced significantly by GABAergic inhibition. AMPA receptors decrease spatial phase-selective simple cell responses and generate phase-invariant complex cell responses.

The role of nitric oxide in the transformation of visual information within the dorsal lateral geniculate nucleus of the cat.

We have shown that application of an inhibitor of the enzyme nitric oxide synthase (NOS) effectively suppresses the visual responses of relay cells in the dorsal lateral geniculate nucleus (dLGN) of the anaesthetized paralysed cat. Such suppression seems to result from a specific reduction in transmission via N-methyl-D-aspartic acid (NMDA) receptors, since iontophoretic application of the inhibitor of NOS selectively and in a dose-dependent manner decreased the responses to exogenously applied NMDA. Responses to other exogenously applied amino acid agonists, such as quisqualate (Quis), kainate (Kain) and alpha-amino-3-hydroxy-5-5-methyl-4-isoxazole-propionic acid (AMPA) were largely unaffected. Furthermore, the excitatory action of acetylcholine (ACh), normally co-localized with NOS in axonal terminals within the dLGN arising from the brainstem, was also unaffected. Unlike some other actions of nitric oxide (NO), this role seems not to involve an increase in production of cyclic guanosine-3',5'-mono-phosphate (cGMP), since application of the membrane permeable cGMP analogue 8-bromo-cGMP did not alter the suppressive effect of NOS inhibitors on either visual or NMDA evoked responses. We conclude that the normal function of NO at this level of the visual system is permissive, allowing full expression of NMDA mediated visually elicited information.

A possible role for nitric oxide at the sleep/wake interface.

Cholinergic neurotransmission is known to have important arousal/activating functions. The neurons responsible for those actions also release the atypical neuromodulator nitric oxide (NO), which has been shown in previous studies to be involved in the modulation of sleep/wake states. The present investigation, using an animal model (anesthetized cat) tests the hypothesis that NO cooperates with ACh in controlling rhythmic neuronal activity, which may play a role in sleep/wake transition. We have used extracellular singleunit recording of neurons in the dorsal thalamus and visual cortex with simultaneous iontophoretic application of drugs acting upon the NO system: the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine (L-NOArg), NO donors, and 8-bromo-cGMP (which mimics the action of NO). Local inhibition of NOS significantly reduced the activity of recorded cells in both thalamus and visual cortex. The opposite effect was achieved with NO donors application. In cortex, ejection of 8-bromo-cGMP or the NO donor diethylamine-nitric oxide (DEA-NO) increased cell firing. Furthermore, the rhythmic firing pattern present in these cortical neurons was disrupted. Taken together, these findings suggest that the NO system collaborates with cholinergic neurotransmission. This collaboration might be involved in the control of different patterns of electrogenic activity during various states of the sleep-wake cycle, via the ability of the NO system to modify rhythmic activity of neurons.

Enhanced visual responses in cat dLGN--potentiation by priming with excitatory amino acids.

Sustained iontophoresis of NMDA potentiated visual responses for minutes after the application in 16 of 38 cells (42%), peaking 3 min after the end of the application and declining to control levels within 12 min. Potentiation was also seen after application of ACPD (36%, n = 14) and AMPA (29%, n = 14), but not after application of ACh (n = 20). ACh also excites dLGN cells, but does not interact with amino acid receptors, and ACh receptors are not directly involved in the transmission of visual information. We suggest that this modulation is a form of visually induced potentiation which permits dynamic modification of the strength of visual information to be relayed to the cortex depending upon the history of previous activity levels.

An unusual effect of application of the amino acid L-arginine on cat visual cortical cells.

Iontophoretic application of L-arginine (L-Arg) resulted in a profound decrease in visually elicited and spontaneous activity in 22 of 77 (29%) cells in area 17 of the anaesthetized/paralysed cat. Duration was long, and cells did not recover pre-application activity levels, indicating permanent decline. This effect was obtained without change in the extracellularly recorded wave-form, demonstrating that this did not result from depolarization block. In the remaining 55 cells, application of L-Arg alone, at levels capable of eliciting inhibition as described above, was without effect. In 29 cells, L-Arg application was able to reverse the effect of inhibition of nitric oxide (NO) production. Populations of cells showing the depressive effect described above and those affected by NO modulation levels were mutually exclusive.

Anandamide activation of CB1 receptors increases spontaneous bursting and oscillatory activity in the thalamus.

The endocannabinoid system is a modulatory system that has been strongly associated with the regulation of functions as learning and memory, pain perception and sensory physiology in many areas of the central nervous system. However, although a role in sensory processing has been demonstrated at the level of the thalamus, the influence of the endocannabinoid system on thalamic rhythms and oscillations has been less studied, despite the fact that such activities are significant characteristics of the thalamic state. The present work aimed to characterize the role of anandamide (AEA) - one of the endogenous CB1 receptor agonists - and AM251 - a CB1 antagonist - in the modulation of burst firing and oscillatory activity present in the dLGN of the anesthetized rat. Administration of AEA (0.5mg/kg iv) increased the number of bursts in the majority of the cells tested and induced the appearance of a slow delta-like (1.5Hz) oscillatory activity. These effects were CB1-mediated, as demonstrated by the complete antagonism during the co-application of AM251 (0.5mg/kg iv). Thus, by demonstrating that the AEA-mediated activation of CB1 receptors increases spontaneous bursting and oscillatory activity in the thalamus our study infers that endocannabinoids could have a role in processes controlling the sleep-wake cycle and level of arousal.

Foci of orientation plasticity in visual cortex.

Cortical areas are generally assumed to be uniform in their capacity for adaptive changes or plasticity. Here we demonstrate, however, that neurons in the cat striate cortex (V1) show pronounced adaptation-induced short-term plasticity of orientation tuning primarily at specific foci. V1 neurons are clustered according to their orientation preference in iso-orientation domains that converge at singularities or pinwheel centres. Although neurons in pinwheel centres have similar orientation tuning and responses to those in iso-orientation domains, we find that they differ markedly in their capacity for adaptive changes. Adaptation with an oriented drifting grating stimulus alters responses of neurons located at and near pinwheel centres to a broad range of orientations, causing repulsive shifts in orientation preference and changes in response magnitude. In contrast, neurons located in iso-orientation domains show minimal changes in their tuning properties after adaptation. The anisotropy of adaptation-induced orientation plasticity is probably mediated by inhomogeneities in local intracortical interactions that are overlaid on the map of orientation preference in V1.

Visual response augmentation in cat (and macaque) LGN: potentiation by corticofugally mediated gain control in the temporal domain.

Visual responses of neurons are dependent on the context of a stimulus, not only in spatial terms but also temporally, although evidence for temporally separate visual influences is meagre, based mainly on studies in the higher cortex. Here we demonstrate temporally induced elevation of visual responsiveness in cells in the lateral geniculate nucleus (LGN) of cat and monkey following a period of high intensity (elevated contrast) stimulation. This augmentation is seen in 40-70% (monkey-cat) of cells tested and of all subtypes. Peaking at approximately 3 min following the period of intense stimulation, it can last for 10-12 min and can be repeated and summed in time. Furthermore, it is dependent on corticofugal input, is seen even when high contrast stimuli of orthogonal orientation are used and therefore results from a/any prior increase in activity in the retino-geniculo-striate pathway. We suggest that this reflects a general mechanism for control of visual responsiveness; both a flexible and dynamic means of changing effectiveness of thalamic activity as visual input changes, but also a mechanism which is an emergent property of the thalamo-cortico-thalamic loop.

Depth perception in random dot stereograms is not affected by changes in either vergence or accommodation.

PURPOSE: To test the hypothesis that extraretinal cues related to vergence angle and lens accommodation are used to scale horizontal disparities for fixation distance. METHODS: Depth perception of random dot stereograms was studied in 10 healthy adult subjects with normal visual acuity by modifying retinal disparity, fixation distance, vergence angle, and accommodation. Statistical analysis was used to compare the data. RESULTS: Depth perception increased with fixation distance. The increment of depth perception persisted even when horizontal retinal disparity was kept constant. The magnitude of depth perception was independent of vergence angle. Depth perception did not vary with changes in accommodation. CONCLUSIONS: Extraretinal cues related to vergence angle and accommodation seem to be not necessary to scale horizontal disparities for viewing distance.

Modulatory influence of putative inhibitors of nitric oxide synthesis on visual processing in the cat lateral geniculate nucleus.

1. Using an in vivo preparation we have examined the actions of two inhibitors of nitric oxide synthase (NOS), NG-nitro-L-arginine (L-NOArg) and NG-methyl-L-arginine (L-MeArg), in the feline dorsal lateral geniculate nucleus (dLGN). We compared the responses obtained to iontophoretic application of these substances during visual stimulation with those elicited by visual stimulation alone. The effects of concurrent ejection of L-arginine (L-Arg), the normal physiological substrate of NOS, and D-arginine, the inactive isomer, were tested on these responses. 2. Extracellular application of L-NOArg and L-MeArg produced clear and repeatable effects, consisting of substantial reduction in discharge rate without affecting response selectivity, on 94% of tested cells. These effects were prevented by simultaneous application of L-Arg, which when ejected alone produced no change on visual evoked responses. 3. The data suggest that nitric oxide (NO) is necessary for the transmission of the visual input under normal visual stimulation and show a direct involvement of NO in visual information processing at the level of dLGN, suggesting that its contribution to brain mechanisms is more profound than previously thought.

The influence of nitric oxide on perigeniculate GABAergic cell activity in the anaesthetized cat.

We have tested the effect of iontophoretic application of the nitric oxide synthase inhibitor L-nitroarginine on the activity of a population of 53 perigeniculate (PGN) cells, recorded extracellularly in the anaesthetized paralysed cat. In all cells tested with visual stimulation during L-nitroarginine application (n = 15), the visually elicited responses were markedly reduced, on average by 63 +/- 15%, and there was a reduction in spontaneous activity too. This effect was blocked by co-application of the substrate for nitric oxide synthase, L-arginine, but not by the inactive D-isoform, although application of L-arginine alone was without effect. Pressure application of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) elevated both visual responses and spontaneous discharge, an effect also seen with a second nitric oxide donor, sodium nitroprusside (n = 12). The nitric oxide synthase inhibitor L-nitroarginine was applied to a sub-population of seven cells and it selectively decreased NMDA mediated excitation (reduction 80 +/- 14%) with little or no effect on the excitation mediated by alpha-amino-3-hydroxy-5-5-methyl-4-isoxazole-propionic acid (AMPA) or quisqualate (effects not statistically significant), and it had no effect (n = 7) on excitation mediated by the metabotropic agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). Furthermore, application of SNAP also increased the magnitude of excitatory responses mediated by NMDA receptors. On a different population of seven cells, application of the new NO donor diethylamine-nitric oxide (DEA-NO) enhanced the actions of NMDA without an effect on responses to AMPA. These effects are qualitatively and quantitatively similar to those we have previously described for X and Y type cells in the dorsal lateral geniculate nucleus (dLGN), despite the known opposite effects of acetylcholine (ACh) application in the dLGN and PGN (ACh is co-localized with nitric oxide synthase at both sites). We propose that within the PGN nitric oxide acts to enhance transmission utilizing NMDA receptors selectively (thereby interacting with the globally inhibiting effect of ACh at this site) to enhance visual responses, reducing or removing the non-specific inhibitory drive from PGN to dLGN seen in the spindling activity of slow-wave sleep. These effects will act in concert with the facilitatory actions of both ACh and nitric oxide within the dLGN proper, and will thereby enhance the faithful transmission of visual information from retina to cortex.

Effects of feedback projections from area 18 layers 2/3 to area 17 layers 2/3 in the cat visual cortex.

In the absence of a direct geniculate input, area 17 cells in the cat are nevertheless able to respond to visual stimuli because of feedback connections from area 18. Anatomic studies have shown that, in the cat visual cortex, layer 5 of area 18 projects to layer 5 of area 17, and layers 2/3 of area 18 project to layers 2/3 of area 17. What is the specific role of these connections? Previous studies have examined the effect of area 18 layer 5 blockade on cells in area 17 layer 5. Here we examine whether the feedback connections from layers 2/3 of area 18 influence the orientation tuning and velocity tuning of cells in layers 2/3 of area 17. Experiments were carried out in anesthetized and paralyzed cats. We blocked reversibly a small region (300 microm radius) in layers 2/3 of area 18 by iontophoretic application of GABA and recorded simultaneously from cells in layers 2/3 of area 17 while stimulating with oriented sweeping bars. Area 17 cells showed either enhanced or suppressed visual responses to sweeping bars of various orientations and velocities during area 18 blockade. For most area 17 cells, orientation bandwidths remained unaltered, and we never observed visual responses during blockade that were absent completely in the preblockade condition. This suggests that area 18 layers 2/3 modulate visual responses in area 17 layers 2/3 without fundamentally altering their specificity.

Actions of compounds manipulating the nitric oxide system in the cat primary visual cortex.

1. We iontophoretically applied NG-nitro-L-arginine (L-NOArg), an inhibitor of nitric oxide synthase (NOS), to cells (n = 77) in area 17 of anaesthetized and paralysed cats while recording single-unit activity extracellularly. In twenty-nine out of seventy-seven cells (38%), compounds altering NO levels affected visual responses. 2. In twenty-five out of twenty-nine cells, L-NOArg non-selectively reduced visually elicited responses and spontaneous activity. These effects were reversed by co-application of L-arginine (L-Arg), which was without effect when applied alone. Application of the NO donor diethylamine-nitric oxide (DEA-NO) produced excitation in three out of eleven cells, all three cells showing suppression by L-NOArg. In ten cells the effect of the soluble analogue of cGMP, 8-bromo-cGMP, was tested. In three of those in which L-NOArg application reduced firing, 8-bromo-cGMP had an excitatory effect. In six out of fifteen cells tested, L-NOArg non-selectively reduced responses to NMDA and alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA). Again, co-application of L-Arg reversed this effect, without enhancing activity beyond control values. 3. In a further subpopulation of ten cells, L-NOArg decreased responses to ACh in five. 4. In four out of twenty-nine cells L-NOArg produced the opposite effect and increased visual responses. This was reversed by co-application of L-Arg. Some cells were also affected by 8-bromo-cGMP and DEA-NO in ways opposite to those described above. It is possible that the variety of effects seen here could also reflect trans-synaptic activation, or changes in local circuit activity. However, the most parsimonious explanation for our data is that NO differentially affects the activity of two populations of cortical cells, in the main causing a non-specific excitation.

Further observations on the role of nitric oxide in the feline lateral geniculate nucleus.

We have examined the responses of a population of 77 cells in the dorsal lateral geniculate nucleus (dLGN) of the anaesthetized, paralysed cat. Here the synthetic enzyme for the production of nitric oxide, nitric oxide synthase, is found only in the presynaptic terminals of the cholinergic input from the brainstem. In our hands, iontophoretic application of inhibitors of this enzyme resulted both in significant decreases in visual responses and decreased responses to exogenous application of NMDA, effects which were reversed by coapplication of the natural substrate for nitric oxide synthase, L-arginine, but not the biologically inactive isomer, D-arginine. Nitroprusside and S-nitroso-N-acetylpenicillamine (SNAP), nitric oxide donors, but not L-arginine, were able to increase markedly both spontaneous activity and the responsiveness to NMDA application. Furthermore, SNAP application facilitated visual responses. Responses of cells in animals without retinal, cortical and parabrachial input to the LGN suggest a postsynaptic site of action of nitric oxide. This modulation of the gain of visual signals transmitted to the cortex suggests a completely novel pathway for nitric oxide regulation of function, as yet described only in primary sensory thalamus of the mammalian central nervous system.