Orientation selective stimulation with tetrahedral electrodes of the rat infralimbic cortex to indirectly target the amygdala.

Introduction: Deep brain stimulation (DBS) is a rapidly developing therapeutic intervention with constantly expanding neurological and psychiatric indications. A major challenge for the approach is the precise targeting and limitation of the effect on the desired neural pathways. We have introduced a new approach, orientation selective stimulation (OSS) that allows free rotation of the induced electric field on a plane when using a probe with three parallel electrodes forming an equilateral triangle at the tip. Here, we expand the technique by introducing a tetrahedral stimulation probe that enables adjustment of the primary electric field direction freely at any angle in a 3D space around the stimulating probe. OSS in 3D will enable better targeting of the electric field according to the local brain anatomy. We tested its utility in a rat model of DBS for treatment-resistant depression. The stimulation directed to the subgenual anterior cingulate cortex (sgACC) has yielded dramatic improvement in individual patients suffering from therapy resistant depression, but no consistent benefit in larger series. This failure has been ascribed to the challenging anatomy of sgACC with several crossing neural tracts and individual differences in the local anatomy. Methods: We stimulated infralimbic cortex (IL), the rat analog of sgACC, and recorded local electrical responses in amygdala (AMG) that is monosynaptically connected to IL and plays a central role in emotional states. We further traced AMG-IL connections using a viral vector and tractography using diffusion magnetic resonance imaging (MRI). Finally, we mimicked the clinical situation by delivering sustained 130 Hz stimulation at IL at the most effective field orientation and followed changes in resting-state functional connectivity with IL using functional MRI. To help interpretation of responses in functional connectivity, we stimulated only the left IL, which we did not expect to evoke measurable changes in the rat behavior. Results: The AMG evoked responses depended systematically on the IL stimulation field orientation and yielded the maximum response in near vertical field orientation in accordance with tractography. Sustained 130 Hz stimulation at a field orientation yielding the strongest AMG evoked responses increased functional connectivity between IL and AMG on the stimulation side. Conclusion: These findings suggest that OSS in 3D provides a new approach to optimize the DBS for every individual patient with a single stimulation probe implantation.

Preclinical evaluation of drug treatment options for sleep-related epileptiform spiking in Alzheimer's disease.

Introduction: There are no published data on prospective clinical studies on drug treatment options for sleep-related epileptiform spiking in Alzheimer's disease (AD). Methods: Using video-EEG with hippocampal electrodes in 17 APP/PS1 transgenic male mice we assessed the effects of donepezil and memantine, anti-seizure drugs levetiracetam and lamotrigine, gamma-secretase inhibitor semagacestat, anti-inflammatory minocycline and adenosine receptor antagonist istradephylline on density of cortical and hippocampal spikes during sleep. Results: Levetiracetam decreased the density of hippocampal giant spikes and cortical spikes. Lamotrigine reduced cortical single spikes and spike-wave discharges but dramatically increased hippocampal giant spikes. Memantine increased cortical single spikes and spike-wave discharges dose-dependently. Memantine and istradephylline decreased total sleep time while levetiracetam increased it. Lamotrigine decreased REM sleep duration. Other drugs had no significant effects. Discussion: Levetiracetam appears promising for treating sleep-related epileptiform spiking in AD while lamotrigine should be used with caution. Donepezil at low doses appeared neutral but the memantine effects warrant further studies.

Response of spike-wave discharges in aged APP/PS1 Alzheimer model mice to antiepileptic, metabolic and cholinergic drugs.

Epileptic nonconvulsive spike-wave discharges (SWDs) are commonly seen in amyloid plaque bearing transgenic mice but only rarely in their wild-type littermates. To shed light on their possible treatment options, we assessed the effect of drugs with variable and known mechanisms of action on the occurrence of SWDs in aged APPswe/PS1dE9 mice. The treatments included prototypic antiepileptic drugs (ethosuximide and levetiracetam), donepezil as the typical Alzheimer drug and atropine as an antagonistic effect, GABAB antagonist CGP-35348, and alternate energy substrates beta-hydroxybutyrate (BHB), pyruvate and lactate on the occurrence of SWDs in aged APPswe/PS1dE9 mice. All agents were administered by single intraperitoneal injections at doses earlier documented to be effective and response was assessed by recording 3 h of video-EEG. Atropine at 25 mg/kg significantly decreased SWD occurrence in all behavioral states, and also resulted in altered frequency composition of SWDs and general EEG slowing during sleep. Ethosuximide at 200 mg/kg and levetiracetam at 75 mg/kg effectively suppressed SWDs only during a period of mixed behavioral states, but levetiracetam also increased SWDs in sleep. BHB at 1 g/kg decreased SWDs in sleep, while both pyruvate and lactate at the same dose tended to increase SWD number and total duration. Unexpectantly, donepezil at 0.3 mg/kg CGP-35348 at 100 mg/kg had no effect on SWDs. These findings call for re-evaluation of some prevailing theories on neural circuit alternations that underlie SWD generation and show the utility of APP/PS1 mice for testing potential new treatments for nonconvulsive epileptic activity related to Alzheimer pathology.

Characterization of Epileptic Spiking Associated With Brain Amyloidosis in APP/PS1 Mice.

Epileptic activity without visible convulsions is common in Alzheimer's disease (AD) and may contribute adversely to the disease progress and symptoms. Transgenic mice with amyloid plaque pathology also display epileptic seizures, but those are too infrequent to assess the effect of anti-epileptic treatments. Besides spontaneous seizures, these mice also display frequent epileptic spiking in epidural EEG recordings, and these have provided a means to test potential drug treatment to AD-related epilepsy. However, the origin of EEG spikes in transgenic AD model mice has remained elusive, which makes it difficult to relate electrophysiology with underlying pathology at the cellular and molecular level. Using multiple cortical and subcortical electrodes in freely moving APP/PS1 transgenic mice and their wild-type littermates, we identified several types of epileptic spikes among over 15 800 spikes visible with cortical screw electrodes based on their source localization. Cortical spikes associated with muscle twitches, cortico-hippocampal spikes, and spindle and fast-spindle associated spikes were present equally often in both APP/PS1 and wild-type mice, whereas pure cortical spikes were slightly more common in APP/PS1 mice. In contrast, spike-wave discharges, cortico-hippocampal spikes with after hyperpolarization and giant spikes were seen almost exclusively in APP/PS1 mice but only in a subset of them. Interestingly, different subtypes of spikes responded differently to anti-epileptic drugs ethosuximide and levetiracetam. From the translational point most relevant may be the giant spikes generated in the hippocampus that reached an amplitude up to ± 5 mV in the hippocampal channel. As in AD patients, they occurred exclusively during sleep. Further, we could demonstrate that a high number of giant spikes in APP/PS1 mice predicts seizures. These data show that by only adding a pair of hippocampal deep electrodes and EMG to routine cortical epidural screw electrodes and by taking into account underlying cortical oscillations, one can drastically refine the analysis of cortical spike data. This new approach provides a powerful tool to preclinical testing of potential new treatment options for AD related epilepsy.

PPARß/δ-agonist GW0742 ameliorates dysfunction in fatty acid oxidation in PSEN1ΔE9 astrocytes.

Astrocytes are the gatekeepers of neuronal energy supply. In neurodegenerative diseases, bioenergetics demand increases and becomes reliant upon fatty acid oxidation as a source of energy. Defective fatty acid oxidation and mitochondrial dysfunctions correlate with hippocampal neurodegeneration and memory deficits in Alzheimer's disease (AD), but it is unclear whether energy metabolism can be targeted to prevent or treat the disease. Here we show for the first time an impairment in fatty acid oxidation in human astrocytes derived from induced pluripotent stem cells of AD patients. The impairment was corrected by treatment with a synthetic peroxisome proliferator activated receptor delta (PPARß/δ) agonist GW0742 which acts to regulate an array of genes governing cellular metabolism. GW0742 enhanced the expression of CPT1a, the gene encoding for a rate-limiting enzyme of fatty acid oxidation. Similarly, treatment of a mouse model of AD, the APP/PS1-mice, with GW0742 increased the expression of Cpt1a and concomitantly reversed memory deficits in a fear conditioning test. Although the GW0742-treated mice did not show altered astrocytic glial fibrillary acidic protein-immunoreactivity or reduction in amyloid beta (Aß) load, GW0742 treatment increased hippocampal neurogenesis and enhanced neuronal differentiation of neuronal progenitor cells. Furthermore, GW0742 prevented Aß-induced impairment of long-term potentiation in hippocampal slices. Collectively, these data suggest that PPARß/δ-agonism alleviates AD related deficits through increasing fatty acid oxidation in astrocytes and improves cognition in a transgenic mouse model of AD.

Hippocampal electrical stimulation disrupts associative learning when targeted at dentate spikes.

KEY POINTS: Dentate spikes are fast fluctuations of hilar local-field potentials that take place during rest and are thought to reflect input arriving from the entorhinal cortex to the hippocampus. During dentate spikes, neuronal firing in hippocampal input (dentate gyrus) and output (CA1/CA3) regions is uncoupled. To date, the behavioural significance of dentate spikes is unknown. Here, we provide evidence that disrupting the dentate spike-related uncoupling of the dentate gyrus and the CA1/CA3 subregions for 1 h after training retards associative learning. We suggest dentate spikes play a significant role in memory consolidation. ABSTRACT: Hippocampal electrophysiological oscillations, namely theta and ripples, have been implicated in encoding and consolidation of new memories, respectively. According to existing literature, hippocampal dentate spikes are prominent, short-duration (<30 ms), large-amplitude (∼2-4 mV) fluctuations in hilar local-field potentials that take place during awake immobility and sleep. Interestingly, previous studies indicate that during dentate spikes dentate gyrus granule cells increase their firing while firing of CA1 pyramidal cells are suppressed, thus resulting in momentary uncoupling of the two hippocampal subregions. To date, the behavioural significance of dentate spikes is unknown. Here, to study the possible role of dentate spikes in learning, we trained adult male Sprague-Dawley rats in trace eyeblink classical conditioning. For 1 h immediately following each conditioning session, one group of animals received hippocampal stimulation via the ventral hippocampal commissure (vHC) contingent on dentate spikes to disrupt the uncoupling between the dentate gyrus and the CA1 subregions. A yoked control group was stimulated during immobility, irrespective of brain state, and another control group was not stimulated at all. As a result, learning was impaired only in the group where vHC stimulation was administered contingent on dentate spikes. Our results suggest dentate spikes and/or the associated uncoupling of the dentate gyrus and the CA1 play a significant role in memory consolidation. Dentate spikes could possibly reflect reactivation and refinement of a memory trace within the dentate gyrus triggered by input from the entorhinal cortex.

Amyloid Plaques Show Binding Capacity of Exogenous Injected Amyloid-ß.

Amyloid plaques, although inducing damage to the immediately surrounding neuropil, have been proposed to provide a relatively innocuous way to deposit toxic soluble amyloid-ß (Aß) species. Here we address this hypothesis by exploring spread and absorption of fluorescent Aß to pre-existing amyloid plaques after local application in wild-type mice versus APP/PS1 transgenic mice with amyloid plaques. Local intracortical or intracerebroventricular injection of fluorescently-labeled Aß in APP/PS1 mice with a high plaque density resulted in preferential accumulation of the peptide in amyloid plaques in both conventional postmortem histology and in live imaging using two-photon microscopy. These findings support the contention that amyloid plaques may act as buffers to protect neurons from the toxic effects of momentary high concentrations of soluble Aß oligomers.

Genetic ablation of tenascin-C expression leads to abnormal hippocampal CA1 structure and electrical activity in vivo.

Despite evidence that the extracellular matrix glycoprotein tenascin-C (TNC) is implicated in brain development and plasticity, its roles in the intact adult brain are unknown. Here we report that spontaneous local field potential (LFP) activity in freely moving adult TNC-deficient mice is abnormal. The power of cortical and hippocampal theta and gamma oscillations was enhanced in comparison to wild-type mice. The alteration in hippocampal gamma rhythm was subfield specific, such that CA1 gamma was accentuated while dentate gyrus gamma was normal. Similar to LFP, synaptic transmission and plasticity at perforant path synapses in the dentate gyrus were unaffected by the mutation. Morphological analyses revealed a subfield-specific reduction in the CA1 volume and a reduction in the numbers of somatostatin-positive interneurons in the hippocampus as potential structural substrates of the observed functional aberrations. These findings indicate a role for tenascin-C in structural organization of the CA1 hippocampal subfield and in shaping neural activity.

Aging and alpha-synuclein affect synaptic plasticity in the dentate gyrus.

Although intracellular accumulation of alpha-synuclein (alpha-syn) is a characteristic pathological change in Parkinson's disease, Lewy body dementia and Alzheimer's disease, the normal function of this presynaptic protein is still unknown. To assess the contribution of alpha-syn to synaptic plasticity as well as to age-related synaptic degeneration in mice, we compared adult and aged mice overexpressing mutated (A30P) human alpha-syn with their nontransgenic littermates using behavioral tests and electrophysiological measures in the dentate gyrus. We found decreased basal synaptic transmission and paired-pulse facilitation in the perforant path-dentate granule cell synapses of aged mice. In addition, alpha-syn accumulation in aged A30P mice but not in aged wild-type mice led to long-term depression of synaptic transmission after a stimulation protocol that normally induces long-term potentiation. These findings suggest that overexpression of mutated alpha-syn exacerbates the aging process and leads to impaired synaptic plasticity.

Development of flexible microelectrode arrays for recording cortical surface field potentials.

The fabrication, characterization and application of an 8-channel flexible microelectrode array for recording cortical surface field potentials is described. Polyimide-based microelectrode arrays were fabricated by using photolithography and physical vapour deposition (PVD) methods. Polyimide was chosen as the substrate and insulation material due to its suitable mechanical properties and biocompatibility. Electrodes and transmission lines were formed by sputter-coating of platinum thin films. Microelectrode arrays were characterized and tested successfully in vitro by impedance spectroscopy and in vivo by somatosensory evoked potential (SEP) recordings in rats. These tests indicated good performance and the potential of microelectrode arrays.

Role of alpha-synuclein in synaptic glutamate release.

Defective mobilization of dopamine from the reserve pool has been reported in both alpha-synuclein knockout mice (KO) and pPrp-A30P transgenic mice. The present study extends these findings to glutamate release. Standard hippocampal slices were prepared from KO, pPrp-A30P, and C57BL/6J wild type (WT1) mice, as well as from mice with transgenic overexpression of wild type human alpha-synuclein (pSyn-hASY) and their negative littermates (WT2), and field responses were measured in CA3 in response to mossy fiber stimulation. The input/output curves indicated no differences in basal synaptic transmission between groups. Paired-pulse facilitation was significantly weaker in both transgenic alpha-synuclein lines and KO mice compared to their controls. High-frequency stimulation induced LTP only in transgenic mice. Frequency-facilitation was absent in KO mice and different from other mouse lines. These findings support the idea that lack of alpha-synuclein impairs mobilization of glutamate from the reserve pool. However, transgenic expression of A30P mutated or wild type alpha-synuclein does not appear to prevent endogenous mouse alpha-synuclein to carry out this function.

Increased hippocampal and cortical beta oscillations in mice deficient for the HNK-1 sulfotransferase.

The HNK-1 carbohydrate is detectable in perineuronal nets around inhibitory neurons in the hippocampus and neocortex. To address the functional contribution of HNK-1 to interneuron function in the adult brain, we recorded EEG and auditory-evoked potential in freely moving mice deficient for HNK-1 sulfotransferase (ST-/- mice) and in wild-type littermates. While ST-/- mice displayed normal theta oscillations, both cortical and hippocampal oscillations within the beta range were enhanced, and gamma oscillations showed an opposite trend. ST-/- mice had amplitudes of auditory-evoked potentials similar to control mice, but the latencies of their hippocampal responses were shorter. Morphological analysis revealed a decreased density of parvalbumin-positive interneurons in the hippocampal CA3 subfield of ST-/- mice, which may contribute to the observed changes in networks oscillations. These findings reveal alterations in ST-/- mice that differ from EEG abnormalities of mice deficient in the HNK-1 carrier molecule tenascin-R.

Cognitive aging and the hippocampus: how old rats represent new environments.

Spatial learning impairment in aged rats is associated with changes in hippocampal connectivity and plasticity. Several studies have explored the age-related deficit in spatial information processing by recording the location-specific activity of hippocampal neurons (place cells). However, these studies have generated disparate characterizations of place cells in aged rats as unstable (Barnes et al., 1997), resistant to change (Tanila et al., 1997b; Oler and Markus, 2000; Wilson et al., 2003), or delayed in using external cues (Rosenzweig et al., 2003). To reconcile these findings, we recorded place cells from aged and young rats as they repeatedly explored both a highly familiar environment and an initially novel environment, and we repeatedly tested whether the place fields formed in the novel environment were anchored by external cues. Initially, spatial representations in aged rats were abnormally maintained between the familiar and novel environments. Then, new representations were formed but were also delayed in becoming anchored to the external landmarks. Finally, even when the new spatial representations became bound to the landmarks, they were multi-stable across repetitive exposures to the formerly novel environment. These observations help to reconcile previously divergent characterizations of spatial representation in aged rats and suggest a model of cognitive aging and hippocampal function.

Long-term functional consequences of transient occlusion of the middle cerebral artery in rats: a 1-year follow-up of the development of epileptogenesis and memory impairment in relation to sensorimotor deficits.

Post-stroke seizures occur in 5-20% of patients. Modeling of stroke-induced seizures in animals provides a useful tool for investigating the molecular basis of epileptogenesis and for developing therapies for stroke patients at increased risk for epileptogenesis. The questions addressed in the study were: (1) Do rats develop spontaneous seizures after transient occlusion of the middle cerebral artery (MCAO)? (2) Is epileptogenesis associated with impaired hippocampus-dependent spatial learning and memory? (3) Are the functional abnormalities linked to axonal plasticity in the dentate gyrus? (4) Does the sensorimotor impairment induced by MCAO predict the risk of epileptogenesis? Adult male Sprague-Dawley rats were subjected to MCAO for 120 min. Development of spontaneous seizures was monitored by 1 week of continuous video-electroencephalographic (EEG) recordings at 3, 7, and 12 months after MCAO. Spontaneous seizures were not detected during 1-year follow-up in ischemic rats. Animals were, however, impaired in the spatial memory task (P<0.001), which was not associated with altered hippocampal LTP or abnormal mossy fiber sprouting (Timm staining). Animals also had a long-lasting sensorimotor deficit (P<0.05). The present study indicates that MCAO causes long-lasting sensorimotor and spatial memory impairment, but does not induce epileptogenesis or spontaneous seizures.

Estrogen treatment alleviates NMDA-antagonist induced hippocampal LTP blockade and cognitive deficits in ovariectomized mice.

Estrogen is implicated in hippocampus-dependent spatial learning as well as structural organization and electrophysiological properties of the rat hippocampus but little is known about its mechanisms of action in mice. In this study, we investigated pharmacologically whether estrogen interacts with the hippocampal N-methyl-D-aspartate (NMDA) receptors in ovariectomized mice as postulated for rats. Female C57BL/6J mice were ovariectomized at 5 months, and 2 weeks before testing at 12 months, half of them received subcutaneous estrogen pellets containing 0.18 mg of 17 beta-estradiol. The competitive NMDA-antagonist, 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), was administered at 5.0 and 10.0 microM to block induction of long-term potentiation (LTP) in the hippocampal slice and intraperitoneally at 0.5, 2.0, and 5.0 mg/kg to impair spatial learning in the water maze. Estrogen treatment shifted the dose-response curve to CPP in both experiments. First, 10 microM CPP blocked the initiation of LTP in all mice, but 5 microM only in ovariectomized non-estrogen-treated mice. Second, final level of acquisition and probe trial performance in the water maze were less affected by high doses of CPP in the estrogen-treated ovariectomized mice than in non-treated group. In control tests for motor side effects, estrogen treatment did not reduce the tendency of CPP to decrease locomotor activity in the open field and impair balance on a rotating rod, and estrogen by itself decreased swimming speed as did CPP, but these effects did not interact. Our findings support the notion that estrogen treatment increases the number of active NMDA-receptors in the mouse hippocampus.