Photostimulation activates fast-spiking interneurons and pyramidal cells in the entorhinal cortex of Thy1-ChR2-YFP line 18 mice.

The application of optogenetics in animals has provided new insights into both fundamental neuroscience and diseases of the nervous system. This is primarily due to the fact that optogenetics allows selectively activating or inhibiting particular types of neurons. One of the first transgenic mouse lines developed for the optogenetic experiment was Thy1-ChR2-YFP. Thy1 is an immunoglobulin superfamily member expressing in projection neurons, so it was assumed that channelrhodopsin-2 (ChR2) would be primarily expressed in projection neurons. However, the specificity of ChR2 expression under promoter Thy1 in different lines has to be clarified yet. Therefore, we aimed to determine the cell specificity of ChR2 expression in the entorhinal cortex of Thy1-ChR2-YFP line 18 mice. We have found that both pyramidal cells and fast-spiking interneurons in deep layers of the entorhinal cortex depolarized and fired in response to 470-nm photostimulation. To exclude the effect of synaptic activation of interneurons by pyramidal cells, we used a selective antagonist of AMPA receptors. Under these conditions, inhibitory postsynaptic currents decreased but did not disappear completely. Furthermore, gabazine inhibited these postsynaptic currents entirely, thus confirming the direct activation of interneurons by light. These data demonstrate that ChR2 is expressed in both pyramidal neurons and fast-spiking interneurons of the entorhinal cortex in Thy1-ChR2-YFP mice.

Quantitative Imaging Biomarkers of Damage to Critical Memory Regions Are Associated With Post-Radiation Therapy Memory Performance in Brain Tumor Patients.

PURPOSE: We used quantitative magnetic resonance imaging to prospectively analyze the association between microstructural damage to memory-associated structures within the medial temporal lobe and longitudinal memory performance after brain radiation therapy (RT). METHODS AND MATERIALS: Patients with a primary brain tumor receiving fractionated brain RT were enrolled on a prospective trial (n = 27). Patients underwent high-resolution volumetric brain magnetic resonance imaging, diffusion-weighted imaging, and neurocognitive testing before and 3, 6, and 12 months post-RT. Medial temporal lobe regions (hippocampus; entorhinal, parahippocampal, and temporal pole white matter [WM]) were autosegmented, quantifying volume and diffusion biomarkers of WM integrity (mean diffusivity [MD]; fractional anisotropy [FA]). Reliable change indices measured changes in verbal (Hopkins Verbal Learning Test-Revised) and visuospatial (Brief Visuospatial Memory Test-Revised [BVMT-R]) memory. Linear mixed-effects models assessed longitudinal associations between imaging parameters and memory. RESULTS: Visuospatial memory significantly declined at 6 months post-RT (mean reliable change indices, -1.3; P = .012). Concurrent chemotherapy and seizures trended toward a significant association with greater decline in visuospatial memory (P = .053 and P = .054, respectively). Higher mean dose to the left temporal pole WM was significantly associated with decreased FA (r = -0.667; P = .002). Over all time points, smaller right hippocampal volume (P = .021), lower right entorhinal FA (P = .023), greater right entorhinal MD (P = .047), and greater temporal pole MD (BVMT-R total recall, P = .003; BVMT-R delayed recall, P = .042) were associated with worse visuospatial memory. The interaction between right entorhinal MD (BVMT-R total recall, P = .021; BVMT-R delayed recall, P = .004) and temporal pole FA (BVMT-R delayed recall, P = .024) significantly predicted visuospatial memory performance. CONCLUSIONS: Brain tumor patients exhibited visuospatial memory decline post-RT. Microstructural damage to critical memory regions, including the hippocampus and medial temporal lobe WM, were associated with post-RT memory decline. The integrity of medial temporal lobe structures is critical to memory performance post-RT, representing possible avoidance targets for memory preservation.

Impact of video games on plasticity of the hippocampus.

The hippocampus is critical to healthy cognition, yet results in the current study show that action video game players have reduced grey matter within the hippocampus. A subsequent randomised longitudinal training experiment demonstrated that first-person shooting games reduce grey matter within the hippocampus in participants using non-spatial memory strategies. Conversely, participants who use hippocampus-dependent spatial strategies showed increased grey matter in the hippocampus after training. A control group that trained on 3D-platform games displayed growth in either the hippocampus or the functionally connected entorhinal cortex. A third study replicated the effect of action video game training on grey matter in the hippocampus. These results show that video games can be beneficial or detrimental to the hippocampal system depending on the navigation strategy that a person employs and the genre of the game.

Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons.

Previous studies have revealed that extremely low frequency electromagnetic field (ELF-EMF) exposure affects neuronal dendritic spine density and NMDAR and AMPAR subunit expressions in the entorhinal cortex (EC). Although calcium signaling has a critical role in control of EC neuronal functions, however, it is still unclear whether the ELF-EMF exposure affects the EC neuronal calcium homeostasis. In the present study, using whole-cell recording and calcium imaging, we record the whole-cell inward currents that contain the voltage-gated calcium currents and show that ELF-EMF (50Hz, 1mT or 3mT, lasting 24h) exposure does not influence these currents. Next, we specifically isolate the high-voltage activated (HVA) and low-voltage activated (LVA) calcium channels-induced currents. Similarly, the activation and inactivation characteristics of these membrane calcium channels are also not influenced by ELF-EMF. Importantly, ELF-EMF exposure reduces the maximum amplitude of the high-K(+)-evoked calcium elevation in EC neurons, which is abolished by thapsigargin, a Ca(2+) ATPase inhibitor, to empty the intracellular calcium stores of EC neurons. Together, these findings indicate that ELF-EMF exposure specifically influences the intracellular calcium dynamics of cultural EC neurons via a calcium channel-independent mechanism.

Neuroimaging assessment of memory-related brain structures in a rat model of acute space-like radiation.

PURPOSE: To investigate the acute effects on the central nervous system (CNS) of (56)Fe radiation, a component of high-energy charged particles (HZE) in space radiation, using quantitative magnetic resonance imaging (MRI) noninvasively. MATERIALS AND METHODS: Sprague-Dawley rats were exposed to whole-brain (56)Fe (0, 1, 2, and 4 Gy). At 1 week postirradiation, MRI scans were made using T2-weighted (T2WI), diffusion-weighted (DWI), and contrast enhanced T1-(CET1) imaging. T2 relaxation time and apparent diffusion coefficient (ADC) values were obtained from memory-related brain regions of interest (ROIs). Histopathology was correlated using ex vivo tissues. RESULTS: No overt abnormalities were visualized using T2WI and DWI at 1 week postradiation. CET1 values did not differ significantly between the irradiated and control animals. Compared to 0 Gy, there were significant prolongations in T2 values and reductions in ADC after irradiation. In the absence of evident neuronal pathology, immunohistochemistry revealed astrocytic activation in 4 Gy animals. CONCLUSION: At 1 week after whole-brain (56)Fe exposure, T2 and ADC values can differentiate radiosensitivity in regions critical for hippocampal-related memory. MRI may provide noninvasive assessment of the initial molecular/cellular disturbances in vivo after HZE irradiation.

Inhibition of the betaine-GABA transporter (mGAT2/BGT-1) modulates spontaneous electrographic bursting in the medial entorhinal cortex (mEC).

Disruptions in GABAergic neurotransmission have been implicated in numerous CNS disorders, including epilepsy and neuropathic pain. Selective inhibition of neuronal and glial GABA transporter subtypes may offer unique therapeutic options for regaining balance between inhibitory and excitatory systems. The ability of two GABA transport inhibitors to modulate inhibitory tone via inhibition of mGAT1 (tiagabine) or mGAT2/BGT-1 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-(methylamino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), also known as EF1502) was evaluated using an in vitro model of spontaneous interictal-like bursting (SB). SBs were recorded extracellularly in combined mEC-HC horizontal brain slices (400 microm; 31+/-1 degrees C) obtained from KA-treated rats. Slice recordings demonstrated that EF1502 exhibited a concentration-dependent reduction in SB frequency. EF1502 significantly reduced SB rate to 32% of control at the 30 microM concentration, while reducing the area and duration of SB activity to 60% and 46% of control, respectively, at the 10 microM concentration. In contrast, the GAT1 selective inhibitor tiagabine (3, 10, and 30 microM) was unable to significantly reduce the frequency of SB activity in the mEC, despite significantly reducing both the duration (51% of control) and area (58% of control) of the SB at concentrations as low as 3 microM. The ability of EF1502, but not tiagabine, to inhibit SBs in the mEC suggests that this in vitro model of pharmacoresistant SB activity is useful to differentiate between novel anticonvulsants with similar mechanisms of action and suggests a therapeutic potential for non-GAT1 transport inhibitors.

Loss of synapses in the entorhinal-dentate gyrus pathway following repeated induction of electroshock seizures in the rat.

The goal of this study was to answer the question of whether repeated administration of electroconvulsive shock (ECS) seizures causes structural changes in the entorhinal-dentate projection system, whose neurons are known to be particularly vulnerable to seizure activity. Adult rats were administered six ECS seizures, the first five of which were spaced by 24-hr intervals, whereas the last two were only 2 hr apart. Stereological approaches were employed to compare the total neuronal and synaptic numbers in sham- and ECS-treated rats. Golgi-stained material was used to analyze dendritic arborizations of the dentate gyrus granule cells. Treatment with ECS produced loss of neurons in the entorhinal layer III and in the hilus of the dentate gyrus. The number of neurons in the entorhinal layer II, which provides the major source of dentate afferents, and in the granular layer of the dentate gyrus, known to receive entorhinal projections, remained unchanged. Despite this, the number of synapses established between the entorhinal layer II neurons and their targets, dentate granule cells, was reduced in ECS-treated rats. In addition, administration of ECS seizures produced atrophic changes in the dendritic arbors of dentate granule cells. The total volumes of entorhinal layers II, III, and V-VI were also found to be reduced in ECS-treated rats. By showing that treatment with ECS leads to partial disconnection of the entorhinal cortex and dentate gyrus, these findings shed new light on cellular processes that may underlie structural and functional brain changes induced by brief, generalized seizures.

Spatiotemporal analyses of interactions between entorhinal and CA1 projections to the subiculum in rat brain slices.

The subiculum and the entorhinal cortex (EC) are important structures in processing and transmitting information between the neocortex and the hippocampus. The subiculum potentially receives information from the EC through two routes. In addition to a direct projection from EC to the subiculum, there is an indirect polysynaptic connection. The latter uses a number of possible pathways, which all converge onto the final projection from the hippocampal field CA1 to the subiculum. In this series of experiments we investigated to what extent activity in both pathways influences population activity of subicular neurons. We used voltage sensitive dyes in combined hippocampal-EC slices of the rat to measure the spatio-temporal activity patterns. To activate the two inputs to the subiculum, stimulation electrodes were placed in the stratum oriens/alveus of CA1 and in layer III of the medial EC. The response patterns evoked in the subiculum after electrical stimulation of each of these input pathways separately were compared with the response patterns after simultaneous stimulation of both areas (medial EC + CA1). A comparison of the computed added responses of the two individual stimulations with the measured responses after simultaneous stimulation suggests that both inputs are linearly added in the subiculum with very little nonlinear interactions. This strongly suggests that in the subiculum interaction at a single cell level of the direct and the indirect pathways from the EC is an unlikely scenario.

Expression of the mu-opioid receptor is induced in dentate gyrus granule cells after focal cerebrocortical ischaemia and stimulation of entorhinal afferents.

Focal ischaemia in the cerebral cortex affects the inducibility of long-term potentiation (LTP) in the hippocampus. This impairment of hippocampal function may result from excessive activation of cortico-hippocampal afferents and subsequent perturbation of hippocampal LTP-relevant transmitter systems, which include opioids. Here, we tested if permanent focal ischaemia and electrical afferent stimulation influence the expression of the mu-opioid receptor (MOR) in the rat hippocampus. In the applied ischaemia model, the entire ipsilateral cortical hemisphere and hippocampus experienced sustained excitation as indicated by a long-lasting increase in the expression of arg 3.1/arc (ARG) mRNA, a marker for neuronal activity. Expression of MOR mRNA and protein was strongly increased in granule cells, which contain very low MOR levels under normal conditions, but not in gamma-aminobutyric acid (GABA)ergic neurons, which express the MOR constitutively. In the molecular layer, which contains the dendrites of granule cells, focal ischaemia caused a redistribution of MOR-like immunoreactivity. In contrast to the dentate gyrus, MOR expression was unaltered in the hippocampus proper and in non-infarcted cortical areas. Repetitive high-frequency stimulation of cortico-hippocampal perforant path afferents induced strong MOR mRNA expression throughout the granular layer. However, weak tetanization sufficient to induce LTP and ARG expression did not influence MOR mRNA levels. Taken together, we provide direct evidence for the induction of MOR expression in granule cells experiencing sustained excitation by cortical afferents. In activated, MOR-expressing granule cells, inhibitory opioids may counter-regulate glutamatergic excitation by the perforant path.

The role of adenosine A(1) receptors in the interaction between amygdala and entorhinal cortex of kindled rats.

In this study the effect of adenosine A(1) receptors of the entorhinal cortex (EC) and amygdala on kindled seizures was investigated. Animals were kindled by daily electrical stimulation of amygdala (group 1) or EC (group 2). In the fully kindled animals, N(6)-cyclohexyladenosine (CHA), a selective A(1) receptor agonist, and 1,3-dimethyl-8-cyclopenthylxanthine (CPT), a selective A(1) receptor antagonist, were microinjected bilaterally into the EC (group 1) or amygdala (group 2). The seizure parameters were measured at 5, 15, 60 and 120 min post injection. Obtained data showed that in group 1, intra-EC microinjection of CHA at concentration of 10 microM reduced amygdala- and, EC-afterdischarge duration and stage 5 seizure duration at 5, 15, 60 and 120 min post drug injection. It also increased the latency to stage 4 seizure but no alteration was observed in seizure stage. At concentrations of 0.1 and 1 microM, CHA reduced only EC-afterdischarge duration at 5 and 15 min post drug infusion. Bilateral microinjection CPT at concentrations of 5 and 10 microM into the EC did not alter seizure parameters. Intra-EC microinjection of CPT (5 microM), 5 min before CHA (10 microM), blocked the anticonvulsant effects of CHA. On the other hand, in group 2 animals, intra-amygdala CHA (10, 50 and 100 microM) or CPT (5 and 10 microM) had no significant effect on seizure parameters of EC-kindled rats. These results suggest that adenosine A(1) receptors activation of the EC may have an inhibitory effect on amygdala-kindled seizures. But, despite of reciprocal interconnections between these two regions, activation of the A(1) receptors of the amygdala has no effect on EC-kindled seizures.

Evidence for the perirhinal cortex as a requisite component in the seizure network following seizure repetition in an inherited form of generalized clonic seizures.

Perirhinal cortex (PRh) is strongly implicated in neuronal networks subserving forebrain-driven partial onset seizures, but whether PRh plays a role in generalized onset seizures is unclear. The moderate seizure severity substrain of genetically epilepsy-prone rats (GEPR-3s) exhibits generalized onset clonic audiogenic seizures (AGS), but following repetitive AGS (AGS kindling), an additional behavior, facial and forelimb (F&F) clonus emerges immediately following generalized clonus. F&F clonus is thought to be driven from forebrain structures. The present in vivo study used PRh focal blockade or extracellular PRh neuronal recording with simultaneous behavioral observations to examine the role played by PRh in AGS neuronal networks before and after AGS kindling in GEPR-3s. Bilateral microinjection of an NMDA receptor antagonist [2-amino-7-phosphonoheptanoic acid, AP7 (0.2-7.5 nmol/side)] into PRh did not affect generalized clonus before or after AGS kindling. However, complete and reversible blockade of only the F&F clonic seizure behavior was induced by AP7 (1 and 7.5 nmol) in AGS-kindled GEPR-3s. Significant increases in PRh neuronal responses to acoustic stimuli occurred after AGS kindling. Tonic PRh neuronal firing patterns appeared during generalized clonus before and after AGS kindling. During F&F clonus, burst firing, an indicator of increased excitability, appeared in PRh neurons. These neurophysiological and microinjection findings support a critical role of PRh in generation of this AGS kindling-induced convulsive behavior. These data are the first indication that PRh participates importantly in the neuronal network for AGS as a result of AGS kindling and demonstrate a previously unknown involvement of PRh in generalized onset seizures.

Effects of inescapable stress on LTP in the amygdala versus the dentate gyrus of freely behaving rats.

Stress impairs hippocampal long-term potentiation (LTP), a model of synaptic plasticity that is assumed to underlie memory formation. In the amygdala, little is known about the effects of stress on LTP, or about its longevity. Here we assessed the ability of entorhinal cortex (EC) stimulation to induce LTP simultaneously in the basal amygdaloid nucleus (B) and in the dentate gyrus (DG) of freely behaving Wistar rats. We also tested whether LTP persists over days. Once established, we investigated the effects of acute vs. repeated inescapable stressful experiences on LTP in both structures. Results show that B, like DG, sustained LTP for 7 days. Furthermore, a single exposure to moderate stress facilitated LTP in B but did not affect DG LTP. Stress re-exposure inhibited LTP in DG but only long-lasting LTP (>3 days) in B. Behaviourally, animals exhibited a higher immobility when re-exposed to the stressor than with a single/first exposure. These data support a role for B in memory storage. Furthermore, they support a differential involvement of the amygdala and hippocampus in memory formation and storage depending on the emotional characteristics of the experience.

Distinct mechanisms of bidirectional activity-dependent synaptic plasticity in superficial and deep layers of rat entorhinal cortex.

The entorhinal cortex plays a key role in processing memory information in the brain; superficial layers relay information to, and deep layers receive information from, the hippocampus. The cellular mechanisms of memory are thought to include a number that produce long-term potentiation (LTP) and depression (LTD) of synaptic strength. Our work presents evidence that LTP and LTD occur simultaneously at memory-relevant synapses. We report here that low frequency stimulation generates NMDA receptor-dependent LTD in Wistar rat superficial (layers II and III), and LTP in the deep entorhinal cortex layers (layers V and VI). LTP in deep layers is masked by simultaneously occurring voltage-gated calcium channel-dependent LTD. Our data support a novel mechanism for the sliding-threshold (BCM) model of synaptic plasticity: The sliding thresholds for induction of LTP and LTD in entorhinal cortex deep layers will be driven by the relative activation state of NMDA receptors and voltage-gated calcium channels. The co-expression of LTD and LTP at presynaptic sites in the entorhinal cortex deep layers reveals an intriguing mechanism for differential processing of synaptic information, which may underlie the vast dynamic capacity for information storage by this cortical structure.