Simultaneous excitatory and inhibitory dynamics in an excitable laser.

Neocortical systems encode information in electrochemical spike timings, not just mean firing rates. Learning and memory in networks of spiking neurons is achieved by the precise timing of action potentials that induces synaptic strengthening (with excitation) or weakening (with inhibition). Inhibition should be incorporated into brain-inspired spike processing in the optical domain to enhance its information-processing capability. We demonstrate the simultaneous excitatory and inhibitory dynamics in an excitable (i.e., a pulsed) laser neuron, both numerically and experimentally. We investigate the bias strength effect, inhibitory strength effect, and excitatory and inhibitory input timing effect, based on the simulation platform of an integrated graphene excitable laser. We further corroborate these analyses with proof-of-principle experiments utilizing a fiber-based graphene excitable laser, where we introduce inhibition by directly modulating the gain of the laser. This technology may potentially open novel spike-processing functionality for future neuromorphic photonic systems.

Brain heating induced by near-infrared lasers during multiphoton microscopy.

Two-photon imaging and optogenetic stimulation rely on high illumination powers, particularly for state-of-the-art applications that target deeper structures, achieve faster measurements, or probe larger brain areas. However, little information is available on heating and resulting damage induced by high-power illumination in the brain. In the current study we used thermocouple probes and quantum dot nanothermometers to measure temperature changes induced by two-photon microscopy in the neocortex of awake and anaesthetized mice. We characterized heating as a function of wavelength, exposure time, and distance from the center of illumination. Although total power is highest near the surface of the brain, heating was most severe hundreds of micrometers below the focal plane, due to heat dissipation through the cranial window. Continuous illumination of a 1-mm(2) area produced a peak temperature increase of ∼1.8°C/100 mW. Continuous illumination with powers above 250 mW induced lasting damage, detected with immunohistochemistry against Iba1, glial fibrillary acidic protein, heat shock proteins, and activated caspase-3. Higher powers were usable in experiments with limited duty ratios, suggesting an approach to mitigate damage in high-power microscopy experiments.

High-performance genetically targetable optical neural silencing by light-driven proton pumps.

The ability to silence the activity of genetically specified neurons in a temporally precise fashion would provide the opportunity to investigate the causal role of specific cell classes in neural computations, behaviours and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate powerful, safe, multiple-colour silencing of neural activity. The gene archaerhodopsin-3 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in the mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. Furthermore, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green light-drivable proton pump from the fungus Leptosphaeria maculans (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue versus red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of 'optogenetic' voltage and ion modulator, which will broadly enable new neuroscientific, biological, neurological and psychiatric investigations.

Layer-specific modulation of neuronal excitability by 660-nm laser irradiation in mouse neocortex.

It has been reported that laser light irradiation (LLI) could regulate neuronal activities in the forebrain, but little is known if and how LLI in the red wavelength range affects neuronal excitability. Here, we investigated the effects of a continuous diode laser at 660 nm on intrinsic membrane properties and excitability of presumed pyramidal neurons in the thalamocortical input layer (layer 3/4) and in layer 5 of mouse primary auditory cortex using the whole-cell patch-clamp recording technique. In layer 3/4 neurons, 660-nm laser irradiation (LLI-660) at 20 mW for 5 min gradually increased resting membrane potentials, which reached a plateau after irradiation. Concomitantly, LLI-660 decreased onset latency of first action potentials (spikes) without changing spike threshold or peak amplitude, but increased inter-spike interval of initial bursting spike doublets and their peak amplitude ratio. None of these changes was observed in layer 5 neurons. Instead, LLI-660 at 20 mW rapidly reduced spike width ~5 % within 1 min of irradiation onset. The magnitude of this reduction did not change during 5 or 10 min irradiation, and returned quickly to at least baseline levels after turning the LLI off. Decreasing laser power to 10 mW reduced spike width to a lesser extent, suggesting laser power dependence of this phenomenon. These data suggest that LLI-660 regulates different aspects of neuronal excitability in cortical neurons in a layer-dependent manner possibly by affecting different voltage-gated ion channels.

[Lipids in nuclei of neocortex neurons and glia under CNS-syndrome in rats].

The effect of a local exposure of rat heads to X-ray radiation at a dose of 200 Gy on the number of phospho- lipids and neutral lipids in the nuclear fraction ofneocortex neurons and glia has been investigated A decrease in the amount ofphosphatidylinositol and an increase in sphingomyelin in neuronal nuclei occurred 2 h after irradiation at the time of repair of locomotive disorders. The amount of phosphatidylcholine and phosphati- dylinositol dropped, and the amount of sphingomyelin and cholesterol increased in the nuclei ofglial cells of the neocortex. Sphingomyelin, phosphatidylinositol, phosphatidylcholine and cholesterol of neuronal nuclei are involved in the dynamics of the CNS syndrome in mammals. Radio resistance of the responses of lipid nuclei in mammals with the CNS syndrome has been shown and a possible role of lipids in the post-irradia- tion DNA repair has been suggested.

[Experimental investigation of the neuromediator and water-ion metabolism state under the magnet-laser influence].

There were studied in experimental investigations the changes of dophamin synthesis in culture of neurons from middle brain (MB) in a newborn rats as well as in the water-ion metabolism in tissues of the rabbits big brain hemispheres and ultrastructure changes in the rabbits synaptic apparatus of the neocortex and MB neurons under the magnet-laser influence (MLI). The signs of intensive synthesis and transport of dophamin, changes of quantitative indices of water-ion metabolism as well as ultrastructural components in synaptic apparatus of neurons have had witnessed about activation of the neuromediator and water-ion metabolism and the MLI. All the abovementioned substantiates the possibility of MLI application in neurosurgery, neurology, traumatology in states, which are accompanied by disorders of the neuromediator and water-ion metabolism for prophylaxis of possible complications and the patients' quality of life improvement.

Layer I as a putative neurogenic niche in young adult guinea pig cerebrum.

A considerable number of cells expressing typical immature neuronal markers including doublecortin (DCX+) are present around layer II in the cerebral cortex of young and adult guinea pigs and other larger mammals, and their origin and biological implication await further characterization. We show here in young adult guinea pigs that these DCX+ cells are accompanied by in situ cell division around the superficial cortical layers mostly in layer I, but they co-express proliferating cell nuclear antigen (PCNA) and an early neuronal fate determining factor, PAX6. A small number of these DCX+ cells also colocalize with BrdU following administration of this mitotic indicator. Cranial X-ray irradiation causes a decline of DCX+ cells around layer II, and novel environmental exploration induces c-Fos expression among these cells in several neocortical areas. Together, these data are compatible with a notion that DCX+ cortical neurons around layer II might derive from proliferable neuronal precursors around layer I in young adult guinea pig cerebrum, and that these cells might be modulated by experience under physiological conditions.

[Lipids of neocortex after X-ray irradiation of the rat head at a dose of 200 Gy].

X-ray irradiation at a dose of 200 Gy with local exposure of the rat head induced the change of the lipid content in the neocortex tissue. The amount of phosphatidylinositol was decreased, the amount of free fatty acids, diglycerols, sphingomyelin was increased, and the amount of cholesterol had a growth trend in 2 h after X-ray exposition. The results testify in favor of participation of phosphatidylinositol- and sphingomyelin-relating signal systems and cholesterol in early stages of the cerebral radiation syndrome. We suggest that the change of the lipid content in early periods after the effect of a super-high dose of X-ray irradiation indicates the lipid dependence in the elimination of motion damages and the restoration of the functions of nerve cells. Effects on the lipid metabolism in the nerve tissue are promising for correcting the cerebral radiation syndrome.

Spatiotemporal constraints on optogenetic inactivation in cortical circuits.

Optogenetics allows manipulations of genetically and spatially defined neuronal populations with excellent temporal control. However, neurons are coupled with other neurons over multiple length scales, and the effects of localized manipulations thus spread beyond the targeted neurons. We benchmarked several optogenetic methods to inactivate small regions of neocortex. Optogenetic excitation of GABAergic neurons produced more effective inactivation than light-gated ion pumps. Transgenic mice expressing the light-dependent chloride channel GtACR1 produced the most potent inactivation. Generally, inactivation spread substantially beyond the photostimulation light, caused by strong coupling between cortical neurons. Over some range of light intensity, optogenetic excitation of inhibitory neurons reduced activity in these neurons, together with pyramidal neurons, a signature of inhibition-stabilized neural networks ('paradoxical effect'). The offset of optogenetic inactivation was followed by rebound excitation in a light dose-dependent manner, limiting temporal resolution. Our data offer guidance for the design of in vivo optogenetics experiments.

[The effect of X-rays and artificial hypotermia on lipids in rat neocortex].

Lipid content of tissue and of fraction of microsomes in neocortex of Wistar rats was studies under artificial hypothermia, after X-ray irradiation in dose 8 Gy under conditions of normothermia and artificial hypothermia in 48 h. The condition of artificial hypothermia get by cooling of rats to 15-18 degrees C. It was shown, that in fraction of microsomes of hypothermia rats the content of phosphatidylinositol was decreased, and in 48 h after cooling of rats the amount of protein, total and individual phospholipids was increased. The lipid content in tissue and in fraction of microsomes of rats, which were irradiated in normotermia, had no changes after 48 h. In fraction of microsomes of rats, which were irradiated after hypothermia, the amount of protein, total phospholipids, sphingomyelin, phosphatidylcholine and phosphatidylserine is increased trustworthy. Thus, we think, that radioprotective effect of hypotermia may be connected with the accumulation of proteins and of phospholipids in the endoplasmic reticulum membranes of neocortex.

Irradiation exacerbates cortical cytopathology in the Eker rat model of tuberous sclerosis complex, but does not induce hyperexcitability.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by multi-organ pathologies. Most TSC patients exhibit seizures, usually starting in early childhood. The neuropathological hallmarks of the disease - cortical tubers, containing cytopathological neuronal and glial cell types - appear to be the source of seizure initiation. However, the contribution of these aberrant cell populations to TSC-associated epilepsies is not fully understood. To gain further insight, investigators have attempted to generate animal models with TSC-like brain abnormalities. In the current study, we focused on the Eker rat, in which there is a spontaneous mutation of the TSC2 gene (TSC2+/-). We attempted to exacerbate TSC-like brain pathologies with a "second-hit" strategy - exposing young pups to ionizing irradiation of different intensities, and at different developmental timepoints (between E18 and P6). We found that the frequency of occurrence of dysmorphic neurons and giant astrocytes was strongly dependent on irradiation dose, and weakly dependent on timing of irradiation in Eker rats, but not in irradiated normal controls. The frequency of TSC-like pathology was progressive; there were many more abnormal cells at 3 months compared to 1 month post-irradiation. Measures of seizure propensity (flurothyl seizure latency) and brain excitability (paired-pulse and post-tetanic stimulation studies in vitro), however, showed no functional changes associated with the appearance of TSC-like cellular abnormalities in irradiated Eker rats.

Dopamine release in human neocortical slices: characterization of inhibitory autoreceptors and of nicotinic acetylcholine receptor-evoked release.

The autoinhibitory control of electrically evoked release of [3H]-dopamine and the properties of that induced by nicotinic receptor (nAChR) stimulation were studied in slices of the human neocortex. In both models [3H]-dopamine release was action potential-induced and exocytotic. The selective dopamine D2 receptor agonist (-)-quinpirole reduced electrically evoked release of [3H]-dopamine, yielding IC50 and I(max) values of 23 nM and 76%, respectively. Also, the effects of several other subtype-selective dopamine receptor ligands confirmed that the terminal dopamine autoreceptor belongs to the D2 subtype. The autoinhibitory feedback control was slightly operative under stimulation conditions of 90 pulses and 3 Hz, with a biophase concentration of endogenous dopamine of 3.6 nM, and was enhanced under blockade of dopamine reuptake. [3H]-dopamine release evoked in an identical manner in mouse neocortical slices was not inhibited by (-)-quinpirole, suggesting the absence of dopamine autoreceptors in this tissue and underlining an important species difference. Also, nAChR stimulation-induced release of [3H]-dopamine revealed a species difference: [3H]-dopamine release was evoked in human, but not in rat neocortical slices. The nAChRs inducing [3H]-dopamine release most probably belong to the alpha3/beta2subtype, according to the potencies and efficacies of subtype-selective nAChR ligands. Part of these receptors may be located on glutamatergic neurons.

Pulsed ultrasound expands the extracellular and perivascular spaces of the brain.

Diffusion within the extracellular and perivascular spaces of the brain plays an important role in biological processes, therapeutic delivery, and clearance mechanisms within the central nervous system. Recently, ultrasound has been used to enhance the dispersion of locally administered molecules and particles within the brain, but ultrasound-mediated effects on the brain parenchyma remain poorly understood. We combined an electron microscopy-based ultrastructural analysis with high-resolution tracking of non-adhesive nanoparticles in order to probe changes in the extracellular and perivascular spaces of the brain following a non-destructive pulsed ultrasound regimen known to alter diffusivity in other tissues. Freshly obtained rat brain neocortical slices underwent sham treatment or pulsed, low intensity ultrasound for 5min at 1MHz. Transmission electron microscopy revealed intact cells and blood vessels and evidence of enlarged spaces, particularly adjacent to blood vessels, in ultrasound-treated brain slices. Additionally, ultrasound significantly increased the diffusion rate of 100nm, 200nm, and 500nm nanoparticles that were injected into the brain slices, while 2000nm particles were unaffected. In ultrasound-treated slices, 91.6% of the 100nm particles, 20.7% of the 200nm particles, 13.8% of the 500nm particles, and 0% of the 2000nm particles exhibited diffusive motion. Thus, pulsed ultrasound can have meaningful structural effects on the brain extracellular and perivascular spaces without evidence of tissue disruption.

Reduced inhibition in an animal model of cortical dysplasia.

Cortical dysplasia has a strong association with epilepsy in humans, but the underlying mechanisms for this are poorly understood. In utero irradiation of rats produces diffuse cortical dysplasia and neuronal heterotopia in the neocortex and hippocampus. Using in vitro neocortical slices, whole-cell patch-clamp recordings were obtained from pyramidal neurons in dysplastic cortex and control neocortex. Spontaneous IPSCs were reduced in amplitude (35%) and frequency (70%) in pyramidal cells from dysplastic cortex. Miniature IPSCs were reduced in frequency (66%) in dysplastic cortex. Two additional measures of cortical inhibition, monosynaptic evoked IPSCs and paired pulse depression of evoked EPSCs, were also impaired in dysplastic cortex. Spontaneous EPSCs were increased in amplitude (42%) and frequency (77%) in dysplastic cortex, but miniature EPSCs were not different between the two groups. These data demonstrate significant physiological impairment in inhibitory synaptic transmission in experimental cortical dysplasia. This supports previous immunohistochemical findings in this model and observations in humans of a reduction in the density of inhibitory interneurons in dysplastic cortex.

Neocortical long-term potentiation and long-term depression: site of expression investigated by infrared-guided laser stimulation.

The synaptic site of expression of long-term potentiation (LTP) and long-term depression (LTD) is still a matter of debate. To address the question of presynaptic versus postsynaptic expression of neocortical LTP and LTD in a direct approach, we measured the glutamate sensitivity of apical dendrites of layer 5 pyramidal neurons during LTP and LTD. We used infrared-guided laser stimulation to release glutamate from its "caged" form with high spatial and temporal resolution. Responses to photolytically released glutamate and synaptically evoked EPSPs were recorded with patch-clamp pipettes from the neuronal somata. LTP and LTD could be induced by electrical stimulation at the same synapses in succession. The NMDA receptor-dependent LTD was accompanied by a decrease in the dendritic glutamate sensitivity, suggesting a postsynaptic expression of neocortical LTD. In contrast, LTP was never accompanied by a change in the dendritic glutamate sensitivity. A possible explanation for this finding is a presynaptic expression of neocortical LTP. Another set of experiments corroborated these results: Photolytic application of glutamate with a frequency of 5 Hz caused a long-lasting Ca2+ and NMDA receptor-dependent decrease in the dendritic glutamate sensitivity. In contrast, LTP of dendritic glutamate sensitivity was never induced by photostimulation, despite several experimental modifications to prevent washout of the induction mechanism and to induce a stronger postsynaptic Ca2+ influx. In conclusion, our findings provide strong evidence for a postsynaptic expression of neocortical LTD and favor a primarily presynaptic locus of neocortical LTP.

Lateralized neocortical control of T lymphocyte export from the thymus I. Increased export after left cortical stimulation in behaviorally active rats, mediated by sympathetic pathways in the upper spinal cord.

Electrical stimulation of left temporo-parieto-occipital (TPO) cortex in adult male Wistar rats during their behaviorally active phase (nighttime) transiently increased circulating levels of CD4+ and CD8+ T lymphocytes. Comparable stimulation of this cortex on the right decreased circulating levels of these cells. Responses to left or right cortical stimulation were diminished or absent in behaviorally inactive rats (daytime). Since blood glucocorticoid levels were similar before and after left or right stimulation, they did not appear to account for the lateralized changes observed. These lateralized effects were mediated by spinal cord autonomic pathways emerging at Tl-T7 levels. In adult thymectomized rats, CD4+ and CD8+ T cells failed to increase after left sided stimulation. The results suggest that lateralized cerebral cortical functions can acutely and differentially influence blood T cell subset numbers. The results demonstrate a direct neocortical influence on thymic export of mature T cells, mediated by the sympathetic nervous system.

WIN 55,212-2 decreases the spatial spread of neocortical excitation in vitro.

The 'intrinsic optical signal' was used to monitor neuronal network excitability. The cannabinoid receptor type 1 agonist WIN 55,212-2 reduced the intensity and the spatial spread of the intrinsic optical signal and prolonged its kinetics in the rat neocortex in vitro. These effects were antagonized by the cannabinoid receptor antagonist SR141716A. Thus, our results suggest that neocortical network activity is modulated via the activation of cannabinoid receptors. The decrease of neocortical network excitability in the present study is probably due to a decreased excitability of glutamatergic neurons.

Electric cortical stimulation suppresses epileptic and background activities in neocortical epilepsy and mesial temporal lobe epilepsy.

OBJECTIVE: To evaluate the suppressive effect of electric cortical stimulation upon the seizure onset zone and the non-epileptic cortex covered by subdural electrodes in patients with neocortical epilepsy and mesial temporal lobe epilepsy (MTLE). METHODS: Four patients with medically intractable focal epilepsy had implanted subdural electrodes for preoperative evaluation. Cortical functional mapping was performed by intermittently repeating bursts of electric stimulation, which consisted of 50 Hz alternating square pulse of 0.3 ms duration, 1-15 mA, within 5 s. The effect of this stimulation on the seizure onset zones and on the non-epileptic areas was evaluated by comparing spike frequency and electrocorticogram (ECoG) power spectra before and after stimulation. A similar comparison was performed in stimulation of 0.9 Hz of the seizure onset zones for 15 min. RESULTS: When the seizure onset zone was stimulated with high frequency, spike frequency decreased by 24.7%. Logarithmic ECoG power spectra recorded at stimulated electrode significantly decreased in 10-32 Hz band by high frequency stimulation of the seizure onset zone, and in 14-32 Hz band by high frequency stimulation of the non-epileptic area. Low frequency stimulation of the seizure onset zone produced 18.5% spike reduction and slight power decrease in 12-14 Hz. CONCLUSIONS: Both high and low frequency electric cortical stimulation of the seizure onset zone have a suppressive effect on epileptogenicity. Reduction of ECoG fast activities after electric cortical stimulation suggests the augmentation of inhibitory mechanisms in human cortex.

Sensitivity to radiation-induced apoptosis and neuron loss declines rapidly in the postnatal mouse neocortex.

Therapeutic brain irradiation can cause progressive decline in cognitive function, particularly in children, but the reason for this effect is unclear. The study explored whether age-related differences in apoptotic sensitivity might contribute to the increased vulnerability of the young brain to radiation. Postnatal day 1 (P1) to P30 mice were treated with 0-16 Gy whole-body X-irradiation. Apoptotic cells were identified and quantified up to 48 h later using the TdT-UTP nick end-labelling method (TUNEL) and immunohistochemistry for activated caspase-3. The number of neuron-specific nuclear protein (NeuN)-positive and -negative cells were also counted to measure neuronal and non-neuronal cell loss. Significantly greater TUNEL labelling occurred in the cortex of irradiated P1 animals relative to the other age groups, but there was no difference among the P7, P14 and P30 groups. Irradiation decreased the %NeuN-positive cells in the mice irradiated on P1, whereas in P14 animals, irradiation led to an increase in the %NeuN-positive cells. These data demonstrate that neocortical neurons of very young mice are more susceptible to radiation-induced apoptosis. However, this sensitivity decreases rapidly after birth. By P14, acute cell loss due to radiation occurs primarily in non-neuronal populations.

Metabotropic glutamate receptors contribute to neocortical synaptic plasticity in vivo.

Metabotropic glutamate receptors (mGluRs) have been shown to be important for hippocampus-dependent memory, as well as activity-dependent synaptic plasticity in the hippocampus. In this study, we examined the role of mGluRs in the induction of two forms of activity-dependent synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD), in the neocortex of awake, freely-moving rats. The mGluR antagonist AIDA was administered during the induction of LTP or LTD in the motor cortex. There was a 50% reduction of LTP induced in the early component of the evoked response, but there was no effect on the late component and no effect on the induction of LTD. Thus, mGluRs contribute to at least one form of activity dependent synaptic plasticity in the neocortex.