A VALIDATED ANALYTICAL HPLC METHOD FOR THE QUANTIFICATION OF LINCOMYCIN HYDROCHLORIDE IN BULK AND SOLID DOSAGE FORM

Objective: Develop a simple isocratic reverse phase high performance liquid chromatographic method (RP-HPLC) and validate for the determination of lincomycin hydrochloride (LMH) in bulk and pharmaceutical preparations.Methods: RP-HPLC quantification was carried out by using fine pack SIL RPC18 column. The mobile phase (methanol: water) was pumped at a flow rate of 1 ml/min in the ratio of 90:10 v/v and the eluents were monitored at 254 nm.Results: The retention time of the drug was 3.73 min and produced at a linear response in the concentration range of 5-25µg/ml. The percentage RSD was found to be below 2%. The LOD and LOQ were found to be 0.854µg/ml and 0.258µg/ml respectively.Conclusion: Validation of the method was performed for precision, accuracy, linearity, ruggedness, specificity and sensitivity to conform to ICH guidelines for valuation for analytical methods

Enhancer-Driven Gene Expression (EDGE) Enables the Generation of Viral Vectors Specific to Neuronal Subtypes

Although a variety of remarkable molecular tools for studying neural circuits have recently beendeveloped, the ability to deploy them in particular neuronal subtypes is limited by the fact that nativepromoters are almost never specific enough. We recently showed that one can generate transgenicmice with anatomical specificity surpassing that of native promoters by combining enhancers uniquelyactive in particular brain regions with a heterologous minimal promoter, an approach we call EDGE(Enhancer-Driven Gene Expression). Here we extend this strategy to the generation of viral (rAAV)vectors, showing that some EDGE rAAVs can recapitulate the specificity of the corresponding trans-genic lines in wild-type animals, even of another species. This approach thus holds the promise ofenabling circuit-specific manipulations in wild-type animals, not only enhancing our understandingof brain function, but perhaps one day even providing novel therapeutic avenues to approach disor-ders of the brainpublishedVersion© 2020 The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Not All That Is Gold Glitters: PV-IRES-Cre Mouse Line Shows Low Efficiency of Labeling of Parvalbumin Interneurons in the Perirhinal Cortex

The wide diversity of cortical inhibitory neuron types populating the cortex allows the assembly of diverse microcircuits and endows these circuits with different computational properties. Thus, characterizing neuronal diversity is fundamental to describe the building blocks of cortical microcircuits and probe their function. To this purpose, the mouse has emerged as a powerful tool to genetically label and manipulate specific inhibitory cell-types in the mammalian brain. Among these cell-types, the parvalbumin-expressing interneuron type (PV-INs) is perhaps the most characterized. Several mouse lines have been generated to target PV-INs. Among these mouse lines, the PV-IRES-Cre lines is the most widely used and demonstrated a high specificity and efficiency in targeting PV-INs in different cortical areas. However, a characterization of the performance across cortical regions is still missing. Here we show that the PV-IRES-Cre mouse line labels only a fraction of PV immunoreactive neurons in perirhinal cortex and other association areas. Our results point to a yet uncharacterized diversity within the PV-INs and emphasize the need to characterize these tools in specific cortical areas.publishedVersio

Association cortical areas in the mouse contain a large population of fast-spiking GABAergic neurons that do not express parvalbumin

GABAergic neurons represent 10–15% of the neuronal population of the cortex but exert a powerful control over information flow in cortical circuits. The largest GABAergic class in the neocortex is represented by the parvalbumin-expressing fast-spiking neurons, which provide powerful somatic inhibition to their postsynaptic targets. Recently, the density of parvalbumin interneurons has been shown to be lower in associative areas of the mouse cortex as compared with sensory and motor areas. Modelling work based on these quantifications linked the low-density of parvalbumin interneurons with specific computations of associative cortices. However, it is still unknown whether the total GABAergic population of association cortices is smaller or whether another GABAergic type can compensate for the low density of parvalbumin interneurons. In the present study, we investigated these hypotheses using a combination of neuroanatomy, mouse genetics and neurophysiology. We found that the GABAergic population of association areas is comparable with that of primary sensory areas, and it is enriched of fast-spiking neurons that do not express parvalbumin and were not accounted for by previous quantifications. We developed an intersectional viral strategy to demonstrate that the population of fast-spiking neurons is comparable across cortical regions. Our results provide quantifications of the density of fast-spiking GABAergic neurons and offers new biological constrains to refine current models of cortical computations.publishedVersio

Inhibitory Connectivity Dominates the Fan Cell Network in Layer II of Lateral Entorhinal Cortex.

Fan cells in layer II of the lateral entorhinal cortex (LEC) form a main component of the projection to the dentate gyrus, CA3 and CA2 of the hippocampal formation. This projection has a counterpart originating from stellate cells in layer II of the medial entorhinal cortex (MEC). Available evidence suggests that the two pathways carry different information, exemplified by a difference in spatial tuning of cells in LEC and MEC. The grid cell, a prominent position-modulated cell type present in MEC, has been postulated to derive its characteristic hexagonal firing pattern from dominant disynaptic inhibitory connections between hippocampal-projecting stellate cells. Given that grid cells have not been described in LEC, we aim to describe the local synaptic connectivity of fan cells, to explore whether the network architecture is similar to that of the MEC stellate cell. Using a combination of in vitro multicell electrophysiological and optogenetic approaches in acute slices from rodents of either sex, we show that excitatory connectivity between fan cells is very sparse. Fan cells connect preferentially with two distinct types of inhibitory interneurons, suggesting disynaptic inhibitory coupling as the main form of communication among fan cells. These principles are similar to those reported for stellate cells in MEC, indicating an overall comparable local circuit architecture of the main hippocampal-projecting cell types in the lateral and medial entorhinal cortex.acceptedVersion© 2018. This is the authors' accepted and refereed manuscript to the article. Locked until 7.5.2019 due to copyright restrictions. The final authenticated version is available online at: http://www.jneurosci.org/content/38/45/971

Local projections of layer Vb-to-Va are more prominent in lateral than in medial entorhinal cortex

The entorhinal cortex, in particular neurons in layer V, allegedly mediate transfer of information from the hippocampus to the neocortex, underlying long-term memory. Recently, this circuit has been shown to comprise a hippocampal output recipient layer Vb and a cortical projecting layer Va. With the use of in vitro electrophysiology in transgenic mice specific for layer Vb, we assessed the presence of the thus necessary connection from layer Vb-to-Va in the functionally distinct medial (MEC) and lateral (LEC) subdivisions; MEC, particularly its dorsal part, processes allocentric spatial information, whereas the corresponding part of LEC processes information representing elements of episodes. Using identical experimental approaches, we show that connections from layer Vb-to-Va neurons are stronger in dorsal LEC compared with dorsal MEC, suggesting different operating principles in these two regions. Although further in vivo experiments are needed, our findings imply a potential difference in how LEC and MEC mediate episodic systems-consolidation.publishedVersio

All-viral tracing of monosynaptic inputs to single birthdate-defined neurons in the intact brain

Neuronal firing patterns are the result of inputs converging onto single cells. Identifying these inputs, anatomically and functionally, is essential to understand how neurons integrate information. Single-cell electroporation of helper genes and subsequent local injection of recombinant rabies viruses enable precise mapping of inputs to individual cells in superficial layers of the intact cortex. However, access to neurons in deeper structures requires more invasive procedures, including removal of overlying tissue. We developed a method that, through a combination of virus injections, allows us to target 4 or fewer hippocampal cells 48% of the time and a single cell 16% of the time in wild-type mice without use of electroporation or tissue aspiration. We identify local and distant monosynaptic inputs that can be functionally characterized; in vivo; . By expanding the toolbox for monosynaptic circuit tracing, this method will help further our understanding of neuronal integration at the level of single cells

Generation of an enhancer-driven gene expression viral tool specific to dentate granule cell-types through direct hippocampal injection

Accurate investigations of neural circuitry require specific genetic access to individual circuit elements, i.e., the myriad neuronal cell-types in the brain. However, native promoters cannot achieve this because while most genes are expressed in the brain, few are expressed in a single neuronal cell-type. We recently used enhancers, the subcomponents of the transcriptional apparatus which tell promoters when and where to express, combined with heterologous minimal promoters to increase specificity of transgene expression, an approach we call Enhancer-Driven Gene Expression (EDGE). As we discuss, EDGE is a marked improvement in specificity over native promoters, but still requires careful anatomical analysis to avoid off-target effects. In this study we present a more complete set of genomic markers from the mouse brain and characterize a novel EDGE viral vector capable of specifically driving expression in distinct subtypes of hippocampal neurons, even though it can express in other cell-types elsewhere. The advent of cell-type specific viral tools in wild-type animals provides a powerful strategy for neural circuit investigation and holds promise for studies using animal models for which transgenic tools are not available.publishedVersio

Selective Survival and Maturation of Adult-Born Dentate Granule Cells Expressing the Immediate Early Gene Arc/Arg3.1

Progenitor cells in the adult dentate gyrus provide a constant supply of neuronal precursors, yet only a small fraction of these cells survive and develop into mature dentate granule cells (DGCs). A major challenge of current research is thus to understand the stringent selection process that governs the maturation and functional integration of adult-born DGCs. In mature DGCs, high-frequency stimulation (HFS) of the perforant path input elicits robust expression of the immediate early gene Arc/Arg3.1, trafficking of its mRNA to dendrites, and local synthesis of the protein necessary for consolidation of long-term potentiation (LTP). Given the synaptic commitment inherent in LTP consolidation, we considered that HFS-evoked expression of Arc could be used to timemap the functional integration of newborn DGCs. Dividing cells were birthmarked by BrdU-labeling at 1, 7, 14, 21, or 28 days prior to induction of LTP and expression of Arc was examined by confocal microscopy. Contrary to expectation, LTP did not induce Arc expression in newborn cells at any age, suggesting they might be refractory to synaptically-evoked Arc expression for at least one month. Importantly, however, spontaneous expression of Arc was detected in BrdU-labeled cells and strongly associated with the survival and maturation of NeuN-positive DGCs. Moreover, Arc expression at the earliest ages (1 and 7 days), clearly precedes the formation of glutamatergic synapses on new neurons. These results suggest an unexpected early role for Arc in adult-born DGCs, distinct from its functions in LTP, LTD, and homeostatic synaptic plasticity