Local hippocampal fast gamma rhythms precede brain-wide hyperemic patterns during spontaneous rodent REM sleep.

Rapid eye movement sleep (REMS) is a peculiar brain state combining the behavioral components of sleep and the electrophysiological profiles of wake. After decades of research our understanding of REMS still is precluded by the difficulty to observe its spontaneous dynamics and the lack of multimodal recording approaches to build comprehensive datasets. We used functional ultrasound (fUS) imaging concurrently with extracellular recordings of local field potentials (LFP) to reveal brain-wide spatiotemporal hemodynamics of single REMS episodes. We demonstrate for the first time the close association between global hyperemic events - largely outmatching wake levels in most brain regions - and local hippocampal theta (6-10 Hz) and fast gamma (80-110 Hz) events in the CA1 region. In particular, the power of fast gamma oscillations strongly correlated with the amplitude of subsequent vascular events. Our findings challenge our current understanding of neurovascular coupling and question the evolutionary benefit of such energy-demanding patterns in REMS function.

Behavioral effects of deep brain stimulation of different areas of the Papez circuit on memory- and anxiety-related functions.

Deep brain stimulation (DBS) has gained interest as a potential therapy for advanced treatment-resistant dementia. However, possible targets for DBS and the optimal stimulation parameters are not yet clear. Here, we compared the effects of DBS of the CA1 sub-region of the hippocampus, mammillothalamic tract, anterior thalamic nucleus, and entorhinal cortex in an experimental rat model of dementia. Rats with scopolamine-induced amnesia were assessed in the object location task with different DBS parameters. Moreover, anxiety-related side effects were evaluated in the elevated zero maze and open field. After sacrifice, we applied c-Fos immunohistochemistry to assess which memory-related regions were affected by DBS. When comparing all structures, DBS of the entorhinal cortex and CA1 sub-region was able to restore memory loss when a specific set of stimulation parameters was used. No anxiety-related side effects were found following DBS. The beneficial behavioral performance of CA1 DBS rats was accompanied with an activation of cells in the anterior cingulate gyrus. Therefore, we conclude that acute CA1 DBS restores memory loss possibly through improved attentional and cognitive processes in the limbic cortex.

Neural activity propagation in an unfolded hippocampal preparation with a penetrating micro-electrode array.

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 µm in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine-binding site of N-methyl-D-aspartate receptor.

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 µM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation.

Spatial olfactory learning facilitates long-term depression in the hippocampus.

Recently, it has emerged that visual spatial exploration facilitates synaptic plasticity at different synapses within the trisynaptic network. Particularly striking is the finding that visuospatial contexts facilitate hippocampal long-term depression (LTD), raising the possibility that this form of plasticity may be important for memory formation. It is not known whether other sensory modalities elicit similar permissive effects on LTD. Here, we explored if spatial olfactory learning facilitates LTD in the hippocampus region of freely behaving rats. Patterned afferent stimulation of the Schaffer collaterals elicited short-term depression (STD) (<1 h) of evoked responses in the Stratum radiatum of the CA1 region. Coupling of this protocol with novel exploration of a spatial constellation of olfactory cues facilitated short-term depression into LTD that lasted for over 24 h. Facilitation of LTD did not occur when animals were re-exposed 1 week later to the same odors in the same spatial constellation. Evaluation of learning behavior revealed that 1 week after the 1st odor exposure, the animals remembered the odors and their relative positions. These data support that the hippocampus can use nonvisuospatial resources, and specifically can use spatial olfactory information, to facilitate LTD and to generate spatial representations. The data also support that a tight relationship exists between the processing of spatial contextual information and the expression of LTD in the hippocampus.

Dual electrophysiological recordings of synaptically-evoked astroglial and neuronal responses in acute hippocampal slices.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

Rapid compensatory changes in the expression of EAAT-3 and GAT-1 transporters during seizures in cells of the CA1 and dentate gyrus.

BACKGROUND: Epilepsy is a neurological disorder produced by an imbalance between excitatory and inhibitory neurotransmission, in which transporters of both glutamate and GABA have been implicated. Hence, at different times after local administration of the convulsive drug 4-aminopyridine (4-AP) we analyzed the expression of EAAT-3 and GAT-1 transporter proteins in cells of the CA1 and dentate gyrus. METHODS: Dual immunofluorescence was used to detect the co-localization of transporters and a neuronal marker. In parallel, EEG recordings were performed and convulsive behavior was rated using a modified Racine Scale. RESULTS: By 60 min after 4-AP injection, EAAT-3/NeuN co-labelling had increased in dentate granule cells and decreased in CA1 pyramidal cells. In the latter, this decrease persisted for up to 180 min after 4-AP administration. In both the DG and CA1, the number of GAT-1 labeled cells increased 60 min after 4-AP administration, although by 180 min GAT-1 labeled cells decreased in the DG alone. The increase in EAAT-3/NeuN colabelling in DG was correlated with maximum epileptiform activity and convulsive behavior. CONCLUSIONS: These findings suggest that a compensatory mechanism exists to protect against acute seizures induced by 4-AP, whereby EAAT-3/NeuN cells is rapidly up regulated in order to enhance the removal of glutamate from the extrasynaptic space, and attenuating seizure activity.

Survival of mossy cells of the hippocampal dentate gyrus in humans with mesial temporal lobe epilepsy.

OBJECT: Hippocampal sclerosis can be identified in most patients with mesial temporal lobe epilepsy (TLE). Surgical removal of the sclerotic hippocampus is widely performed to treat patients with drug-resistant mesial TLE. In general, both epilepsy-prone and epilepsy-resistant neurons are believed to be in the hippocampal formation. The hilar mossy cells of the hippocampal dentate gyrus are usually considered one of the most vulnerable types of neurons. The aim of this study was to clarify the fate of mossy cells in the hippocampus in epileptic humans. METHODS: Of the 19 patients included in this study, 15 underwent temporal lobe resection because of drug-resistant TLE. Four patients were used as controls because they harbored tumors that had not invaded the hippocampus and they had experienced no seizures. Histological evaluation of resected hippocampal tissues was performed using immunohistochemistry. RESULTS: Mossy cells were identified in the control as well as the epileptic hippocampi by using cocaine- and amphetamine-regulated transcript peptide immunohistochemistry. In most cases the number of mossy cells was reduced and thorny excrescences were smaller in the epileptic hippocampi than in controls; however, there was a significant loss of pyramidal cells and a partial loss of granule cells in the same epileptic hippocampi in which mossy cell loss was apparent. The loss of mossy cells could be correlated with the extent of hippocampal sclerosis, patient age at seizure onset, duration of epilepsy, and frequency of seizures. CONCLUSIONS: In many cases large numbers of mossy cells were present in the hilus of the dentate gyrus when most pyramidal neurons of the CA1 and CA3 areas of the Ammon's horn were lost, suggesting that mossy cells may not be more vulnerable to epileptic seizures than the hippocampal pyramidal neurons.