Telencephalic outputs from the medial entorhinal cortex are copied directly to the hippocampus.

Complementary actions of the neocortex and the hippocampus enable encoding and long-term storage of experience dependent memories. Standard models for memory storage assume that sensory signals reach the hippocampus from superficial layers of the entorhinal cortex (EC). Deep layers of the EC on the other hand relay hippocampal outputs to the telencephalic structures including many parts of the neocortex. Here, we show that cells in layer 5a of the medial EC send a copy of their telencephalic outputs back to the CA1 region of the hippocampus. Combining cell-type-specific anatomical tracing with high-throughput RNA-sequencing based projection mapping and optogenetics aided circuit mapping, we show that in the mouse brain these projections have a unique topography and target hippocampal pyramidal cells and interneurons. Our results suggest that projections of deep medial EC neurons are anatomically configured to influence the hippocampus and neocortex simultaneously and therefore lead to novel hypotheses on the functional role of the deep EC.

Fan Cells in Layer 2 of the Lateral Entorhinal Cortex Are Critical for Episodic-like Memory.

Episodic memory requires different types of information to be bound together to generate representations of experiences. The lateral entorhinal cortex (LEC) and hippocampus are required for episodic-like memory in rodents [1, 2]. The LEC is critical for integrating spatial and contextual information about objects [2-6]. Further, LEC neurons encode objects in the environment and the locations where objects were previously experienced and generate representations of time during the encoding and retrieval of episodes [7-12]. However, it remains unclear how specific populations of cells within the LEC contribute to the integration of episodic memory components. Layer 2 (L2) of LEC manifests early pathology in Alzheimer's disease (AD) and related animal models [13-16]. Projections to the hippocampus from L2 of LEC arise from fan cells in a superficial sub-layer (L2a) that are immunoreactive for reelin and project to the dentate gyrus [17, 18]. Here, we establish an approach for selectively targeting fan cells using Sim1:Cre mice. Whereas complete lesions of the LEC were previously found to abolish associative recognition memory [2, 3], we report that, after selective suppression of synaptic output from fan cells, mice can discriminate novel object-context configurations but are impaired in recognition of novel object-place-context associations. Our results suggest that memory functions are segregated between distinct LEC networks.

Effect of reading to preterm infants on measures of cardiorespiratory stability in the neonatal intensive care unit.

OBJECTIVE: To evaluate the impact of parental bedside reading (PR) on cardio-respiratory (CR) stability of preterm infants. METHODS/STUDY DESIGN: Prospective examination of the impact of PR on CR stability in preterm NICU infants. CR data from 3 time points: pre-reading (3 and 1 h before reading), during PR, and post-reading (1 h after reading) were compared. RESULTS: Eighteen infants born at 23-31wks gestation, and 8 to 56 days old, were enrolled. Episodes of oxygen desaturation to <85% were fewer during PR as compared to the pre-reading periods and were fewer with live and maternal PR. CONCLUSION: Preterm infants showed fewer desaturation events less than 85% during PR than prior to reading exposure. This effect persisted up to 1 h after reading exposure. Desaturation events were fewer with live and maternal PR. Voice exposure can be an important way for parents to participate in the care of their preterm infants.

Stellate Cells in the Medial Entorhinal Cortex Are Required for Spatial Learning.

Spatial learning requires estimates of location that may be obtained by path integration or from positional cues. Grid and other spatial firing patterns of neurons in the superficial medial entorhinal cortex (MEC) suggest roles in behavioral estimation of location. However, distinguishing the contributions of path integration and cue-based signals to spatial behaviors is challenging, and the roles of identified MEC neurons are unclear. We use virtual reality to dissociate linear path integration from other strategies for behavioral estimation of location. We find that mice learn to path integrate using motor-related self-motion signals, with accuracy that decreases steeply as a function of distance. We show that inactivation of stellate cells in superficial MEC impairs spatial learning in virtual reality and in a real world object location recognition task. Our results quantify contributions of path integration to behavior and corroborate key predictions of models in which stellate cells contribute to location estimation.

Early Heart Rate Characteristics Predict Death and Morbidities in Preterm Infants.

OBJECTIVES: To determine whether an early heart rate characteristics (HRC) index (HeRO score), measured in the first day and week after birth predicts death and morbidities compared with established illness severity scores. STUDY DESIGN: For all very low birth weight infants in a single neonatal intensive care unit from 2004-2014, the average first day HRC index was calculated within 24 hours of birth (aHRC-24h) and the average first week HRC index within 7 days of birth (aHRC-7d). The Score for Neonatal Acute Physiology (SNAP-II) and Clinical Risk Indicator for Babies (CRIB-II) were calculated when data were available. The aHRC was compared with the SNAP-II and CRIB-II for predicting death, late-onset septicemia, necrotizing enterocolitis, bronchopulmonary dysplasia, severe intraventricular hemorrhage, or severe retinopathy of prematurity. RESULTS: All 4 scores were associated with death and severe intraventricular hemorrhage (P < .01). The OR and 95% CI for every 1-point increase in aHRC for predicting mortality, adjusted for gestational age, was 1.59 (1.25-2.00) for aHRC-24h and 2.61 (1.58-4.33) for aHRC-7d. High aHRC-7d, SNAP-II, and CRIB-II were associated with bronchopulmonary dysplasia (P < .001). High aHRC-7d was associated with late-onset septicemia (P < .05). None of the scores predicted necrotizing enterocolitis or severe retinopathy of prematurity. CONCLUSIONS: HRC assessed in the first day or first week after birth compares favorably to established risk scores to predict death and morbidities in very low birth weight infants.

Molecularly Defined Circuitry Reveals Input-Output Segregation in Deep Layers of the Medial Entorhinal Cortex.

Deep layers of the medial entorhinal cortex are considered to relay signals from the hippocampus to other brain structures, but pathways for routing of signals to and from the deep layers are not well established. Delineating these pathways is important for a circuit level understanding of spatial cognition and memory. We find that neurons in layers 5a and 5b have distinct molecular identities, defined by the transcription factors Etv1 and Ctip2, and divergent targets, with extensive intratelencephalic projections originating in layer 5a, but not 5b. This segregation of outputs is mirrored by the organization of glutamatergic input from stellate cells in layer 2 and from the hippocampus, with both preferentially targeting layer 5b over 5a. Our results suggest a molecular and anatomical organization of input-output computations in deep layers of the MEC, reveal precise translaminar microcircuitry, and identify molecularly defined pathways for spatial signals to influence computation in deep layers.

Toxicoproteomic analysis of pulmonary carbon nanotube exposure using LC-MS/MS.

Toxicoproteomics is a developing field that utilizes global proteomic methodologies to investigate the physiological response as a result of adverse toxicant exposure. The aim of this study was to compare the protein secretion profile in lung bronchoalveolar lavage fluid (BALF) from mice exposed to non-functionalized multi-walled carbon nanotubes (U-MWCNTs) or MWCNTs functionalized by nanoscale Al2O3 coatings (A-MWCNT) formed using atomic layer deposition (ALD). Proteins were identified using liquid chromatography tandem mass spectrometry (LC-MS/MS), and quantified using a combination of two label-free proteomic methods: spectral counting and MS1 peak area analysis. On average 465 protein groups were identified per sample and proteins were first screened using spectral counting and the Fisher's exact test to determine differentially regulated species. Significant proteins by Fisher's exact test (p<0.05) were then verified by integrating the intensity under the extracted ion chromatogram from a single unique peptide for each protein across all runs. A two sample t-test based on integrated peak intensities discovered differences in 27 proteins for control versus U-MWCNT, 13 proteins for control versus A-MWCNT, and 2 proteins for U-MWCNT versus A-MWCNT. Finally, an in-vitro binding experiment was performed yielding 4 common proteins statistically different (p<0.05) for both the in-vitro and in-vivo study. Several of the proteins found to be significantly different between exposed and control groups are known to play a key role in inflammatory and immune response. A comparison between the in-vitro and in-vivo CNT exposure emphasized a true biological response to CNT exposure.

GABAergic projections from the medial septum selectively inhibit interneurons in the medial entorhinal cortex.

The medial septum (MS) is required for theta rhythmic oscillations and grid cell firing in the medial entorhinal cortex (MEC). While GABAergic, glutamatergic, and cholinergic neurons project from the MS to the MEC, their synaptic targets are unknown. To investigate whether MS neurons innervate specific layers and cell types in the MEC, we expressed channelrhodopsin-2 in mouse MS neurons and used patch-clamp recording in brain slices to determine the response to light activation of identified cells in the MEC. Following activation of MS axons, we observed fast monosynaptic GABAergic IPSPs in the majority (>60%) of fast-spiking (FS) and low-threshold-spiking (LTS) interneurons in all layers of the MEC, but in only 1.5% of nonstellate principal cells (NSPCs) and in no stellate cells. We also observed fast glutamatergic responses to MS activation in a minority (<5%) of NSPCs, FS, and LTS interneurons. During stimulation of MS inputs at theta frequency (10 Hz), the amplitude of GABAergic IPSPs was maintained, and spike output from LTS and FS interneurons was entrained at low (25-60 Hz) and high (60-180 Hz) gamma frequencies, respectively. By demonstrating cell type-specific targeting of the GABAergic projection from the MS to the MEC, our results support the idea that the MS controls theta frequency activity in the MEC through coordination of inhibitory circuits.

Atomic layer deposition coating of carbon nanotubes with aluminum oxide alters pro-fibrogenic cytokine expression by human mononuclear phagocytes in vitro and reduces lung fibrosis in mice in vivo.

BACKGROUND: Multi-walled carbon nanotubes (MWCNTs) pose a possible human health risk for lung disease as a result of inhalation exposure. Mice exposed to MWCNTs develop pulmonary fibrosis. Lung macrophages engulf MWCNTs and produce pro-fibrogenic cytokines including interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and osteopontin (OPN). Atomic layer deposition (ALD) is a novel process used to enhance functional properties of MWCNTs, yet the consequence of ALD-modified MWCNTs on macrophage biology and fibrosis is unknown. METHODS: The purpose of this study was to determine whether ALD coating with aluminum oxide (Al2O3) would alter the fibrogenic response to MWCNTs and whether cytokine expression in human macrophage/monocytes exposed to MWCNTs in vitro would predict the severity of lung fibrosis in mice. Uncoated (U)-MWCNTs or ALD-coated (A)-MWCNTs were incubated with THP-1 macrophages or human peripheral blood mononuclear cells (PBMC) and cell supernatants assayed for cytokines by ELISA. C57BL6 mice were exposed to a single dose of A- or U-MWCNTs by oropharyngeal aspiration (4 mg/kg) followed by evaluation of histopathology, lung inflammatory cell counts, and cytokine levels at day 1 and 28 post-exposure. RESULTS: ALD coating of MWCNTs with Al2O3 enhanced IL-1ß secretion by THP-1 and PBMC in vitro, yet reduced protein levels of IL-6, TNF-α, and OPN production by THP-1 cells. Moreover, Al2O3 nanoparticles, but not carbon black NPs, increased IL-1ß but decreased OPN and IL-6 in THP-1 and PBMC. Mice exposed to U-MWCNT had increased levels of all four cytokines assayed and developed pulmonary fibrosis by 28 days, whereas ALD-coating significantly reduced fibrosis and cytokine levels at the mRNA or protein level. CONCLUSION: These findings indicate that ALD thin film coating of MWCNTs with Al2O3 reduces fibrosis in mice and that in vitro phagocyte expression of IL-6, TNF-α, and OPN, but not IL-1ß, predict MWCNT-induced fibrosis in the lungs of mice in vivo.

Production and titering of recombinant adeno-associated viral vectors.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Parvalbumin-positive CA1 interneurons are required for spatial working but not for reference memory.

Parvalbumin-positive GABAergic interneurons in cortical circuits are hypothesized to control cognitive function. To test this idea directly, we functionally removed parvalbumin-positive interneurons selectively from hippocampal CA1 in mice. We found that parvalbumin-positive interneurons are dispensable for spatial reference, but are essential for spatial working memory.

Studying Cerebellar Circuits by Remote Control of Selected Neuronal Types with GABA(A) Receptors.

Although GABA(A) receptor-mediated inhibition of cerebellar Purkinje cells by molecular layer interneurons (MLIs) has been studied intensely at the cellular level, it has remained unclear how this inhibition regulates cerebellum-dependent behaviour. We have implemented two complementary approaches to investigate the function of the MLI-Purkinje cell synapse on the behavioural level. In the first approach we permanently disrupted inhibitory fast synaptic transmission at the synapse by genetically removing the postsynaptic GABA(A) receptors from Purkinje cells (PC-Deltagamma2 mice). We found that chronic disruption of the MLI-Purkinje cell synapse strongly impaired cerebellar learning of the vestibular occular reflex (VOR), presumably by disrupting the temporal patterns of Purkinje cell activity. However, in PC-Deltagamma2 mice the baseline VOR reflex was only mildly affected; indeed PC-Deltagamma2 mice show no ataxia or gait abnormalities, suggesting that MLI control of Purkinje cell activity is either not involved in ongoing motor tasks or that the system compensates for its loss. To investigate the latter possibility we developed an alternative genetic technique; we made the MLI-Purkinje cell synapse selectively sensitive to rapid manipulation with the GABA(A) receptor modulator zolpidem (PC-gamma2-swap mice). Minutes after intraperitoneal zolpidem injection, these PC-gamma2-swap mice developed severe motor abnormalities, revealing a substantial contribution of the MLI-Purkinje cell synapses to real time motor control. The cell-type selective permanent knockout of synaptic GABAergic input and the fast reversible modulation of GABAergic input at the same synapse illustrate how pursuing both strategies gives a fuller view.