Journal: Hippocampus

Article Title: Target-Selectivity of Parvalbumin-Positive Interneurons in Layer II of Medial Entorhinal Cortex in Normal and Epileptic Animals

doi: 10.1002/hipo.22559

Figure Lengend Snippet: a.) Perisomatically-targeting CCK positive basket cells (CCKBCs) selectively target non-perforant path forming calbindin positive (calb+) principal cells while largely avoiding ipsilaterally-projecting reelin positive (reelin+) principal cells that form the perforant path (DG= dentate gyrus). We investigate in this study whether PV containing basket cells (PVBCs) select their targets with respect to long-range projection pattern. b) Since PVBCs do contact both types of principal cell (Varga et al., 2010), it is possible that individual PVBCs are either simultaneously contacting both calb+ and reelin+ principal cells (top), or that there are two subpopulations of PVBCs that are each selective for one principal cell type (bottom). c) This image was reproduced with permission from Varga et al., 2010 Nature Neuroscience, and demonstrates that CCK-immunopositive axon terminals (left panel) selectively surround the somata of some (asterisks) but not all (triangles) principal cells in MEClayerII (principal cells were visualized by GluR2/3 immunopositivity; middle panel). In contrast, axons from PVBCs (right panel) surround the perisomatic region of most principal cells. Scale bar in c = 10μm.

Article Snippet: For all bouton counting experiments, while electron microscopy was not performed to validate the error rate with selection of boutons, we expect that because all data were analyzed as a comparative study between calb + and reelin + cells within individual slices and, when applicable, between epileptic and sham animals in age-matched slices prepared together, the error rate of bouton identification was considered similar for both principal cell populations and between epileptic and age-matched sham control animals. table ft1 table-wrap mode="anchored" t5 caption a7 detection of: experiments antibody antibody manufacturer & catalogue number concentration reelin all primary mouse anti-reelin Millipore monoclonal AB5364 clone G10 1:2,000 secondary Alexa 647 donkey anti-mouse Jackson Immunoresearch 715-605-151 1:500 calbindin all except PV axon tracing primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary Alexa 594 donkey anti-rabbit Jackson Immunoresearch 711-585-152 1:500 calbindin PV cell axon tracing only primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary DyLight 405 donkey anti-rabbit Jackson Immunoresearch 711-475-152 1:500 PV all PV immunostaining primary goat anti-parvalbumin Swant polyclonal antibody number PVG213 1:1,000 secondary Alexa 488 donkey anti-goat Jackson Immunoresearch 705-545-147 1:500 VGLUT3 all VGLUT3 immunostaining primary guinea pig anti-VGLUT3 Chemicon polyclonal antibody, AB5421 1:10,000 secondary Alexa 488 donkey anti-guinea pig Jackson Immunoresearch 706-545-148 1:500 VGAT PV bouton counting only primary guinea pig anti-VGAT Synaptic Systems polyclonal antibody, number 131 004 1:1,000 secondary Alexa 594 donkey anti-guinea-pig Jackson Immunoresearch 706-585-148 1:500 cell fills all except PV axon tracing primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary DyLight 405 streptavidin Jackson Immunoresearch 016-470-084 1:200 cell fills PV cell axon tracing only primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary Alexa 594 streptavidin Jackson Immunoresearch 016-580-084 1:200 Open in a separate window caption a8 Antibodies and other reagents used for cell detection and associated catalogue information See methods and results for descriptions of use.

Techniques:

Journal: Hippocampus

Article Title: Target-Selectivity of Parvalbumin-Positive Interneurons in Layer II of Medial Entorhinal Cortex in Normal and Epileptic Animals

doi: 10.1002/hipo.22559

Figure Lengend Snippet: Antibodies and other reagents used for cell detection and associated catalogue information See methods and results for descriptions of use. Immunogen detected is listed on the leftmost column, along with the primary and secondary antibodies used in specified experiments and concentrations. Manufacturer information contains hyperlinks to websites for the products used in these experiments.

Article Snippet: For all bouton counting experiments, while electron microscopy was not performed to validate the error rate with selection of boutons, we expect that because all data were analyzed as a comparative study between calb + and reelin + cells within individual slices and, when applicable, between epileptic and sham animals in age-matched slices prepared together, the error rate of bouton identification was considered similar for both principal cell populations and between epileptic and age-matched sham control animals. table ft1 table-wrap mode="anchored" t5 caption a7 detection of: experiments antibody antibody manufacturer & catalogue number concentration reelin all primary mouse anti-reelin Millipore monoclonal AB5364 clone G10 1:2,000 secondary Alexa 647 donkey anti-mouse Jackson Immunoresearch 715-605-151 1:500 calbindin all except PV axon tracing primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary Alexa 594 donkey anti-rabbit Jackson Immunoresearch 711-585-152 1:500 calbindin PV cell axon tracing only primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary DyLight 405 donkey anti-rabbit Jackson Immunoresearch 711-475-152 1:500 PV all PV immunostaining primary goat anti-parvalbumin Swant polyclonal antibody number PVG213 1:1,000 secondary Alexa 488 donkey anti-goat Jackson Immunoresearch 705-545-147 1:500 VGLUT3 all VGLUT3 immunostaining primary guinea pig anti-VGLUT3 Chemicon polyclonal antibody, AB5421 1:10,000 secondary Alexa 488 donkey anti-guinea pig Jackson Immunoresearch 706-545-148 1:500 VGAT PV bouton counting only primary guinea pig anti-VGAT Synaptic Systems polyclonal antibody, number 131 004 1:1,000 secondary Alexa 594 donkey anti-guinea-pig Jackson Immunoresearch 706-585-148 1:500 cell fills all except PV axon tracing primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary DyLight 405 streptavidin Jackson Immunoresearch 016-470-084 1:200 cell fills PV cell axon tracing only primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary Alexa 594 streptavidin Jackson Immunoresearch 016-580-084 1:200 Open in a separate window caption a8 Antibodies and other reagents used for cell detection and associated catalogue information See methods and results for descriptions of use.

Techniques: Concentration Assay, Immunostaining

Journal: Hippocampus

Article Title: Target-Selectivity of Parvalbumin-Positive Interneurons in Layer II of Medial Entorhinal Cortex in Normal and Epileptic Animals

doi: 10.1002/hipo.22559

Figure Lengend Snippet: Properties measured include (see Materials and methods section for detailed descriptions): a) hyperpolarizing and depolarizing subthreshold sweeps demonstrating the 1) RMP, 2) Rin (not shown), 3) saghyperpol, 4) τsag, 5) τmembrane, 6) rebound amplitude, 7) sagdepol First spiking trace not shown includes: 8) rheobase (first sweep with spikes, not shown), 9) delay to first spike (not shown), 10) ISI (not shown), b) A single spike with labels for: 11) threshold, 12) spike amplitude, 13) spike halfwidth, c) Trace in (b) zoomed in on the AHP region of the spike to demonstrate: 14) fAHP 15) DAP 16) sAHP. d) Firing patterns of reelin (left, blue) and calbindin (right, red) containing principal cells differed in a number of ways; pictured are examples of regular spiking sweeps from both cell types and e) examples of the first spike from the first spiking depolarizing sweep from reelin (left, blue) and calbindin (right, red) cells. f) Example 3-dimensional plot of three different measured intrinsic properties that differed significantly between all calbindin (red dots) and reelin (blue dots) cells demonstrates clear separation of calbindin and reelin containing cell populations.

Article Snippet: For all bouton counting experiments, while electron microscopy was not performed to validate the error rate with selection of boutons, we expect that because all data were analyzed as a comparative study between calb + and reelin + cells within individual slices and, when applicable, between epileptic and sham animals in age-matched slices prepared together, the error rate of bouton identification was considered similar for both principal cell populations and between epileptic and age-matched sham control animals. table ft1 table-wrap mode="anchored" t5 caption a7 detection of: experiments antibody antibody manufacturer & catalogue number concentration reelin all primary mouse anti-reelin Millipore monoclonal AB5364 clone G10 1:2,000 secondary Alexa 647 donkey anti-mouse Jackson Immunoresearch 715-605-151 1:500 calbindin all except PV axon tracing primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary Alexa 594 donkey anti-rabbit Jackson Immunoresearch 711-585-152 1:500 calbindin PV cell axon tracing only primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary DyLight 405 donkey anti-rabbit Jackson Immunoresearch 711-475-152 1:500 PV all PV immunostaining primary goat anti-parvalbumin Swant polyclonal antibody number PVG213 1:1,000 secondary Alexa 488 donkey anti-goat Jackson Immunoresearch 705-545-147 1:500 VGLUT3 all VGLUT3 immunostaining primary guinea pig anti-VGLUT3 Chemicon polyclonal antibody, AB5421 1:10,000 secondary Alexa 488 donkey anti-guinea pig Jackson Immunoresearch 706-545-148 1:500 VGAT PV bouton counting only primary guinea pig anti-VGAT Synaptic Systems polyclonal antibody, number 131 004 1:1,000 secondary Alexa 594 donkey anti-guinea-pig Jackson Immunoresearch 706-585-148 1:500 cell fills all except PV axon tracing primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary DyLight 405 streptavidin Jackson Immunoresearch 016-470-084 1:200 cell fills PV cell axon tracing only primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary Alexa 594 streptavidin Jackson Immunoresearch 016-580-084 1:200 Open in a separate window caption a8 Antibodies and other reagents used for cell detection and associated catalogue information See methods and results for descriptions of use.

Techniques:

Journal: Hippocampus

Article Title: Target-Selectivity of Parvalbumin-Positive Interneurons in Layer II of Medial Entorhinal Cortex in Normal and Epileptic Animals

doi: 10.1002/hipo.22559

Figure Lengend Snippet: a) Frequency and b) amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from calbindin positive (calb+) and reelin positive (reelin+) principal cells. Consistent with the reduced anatomical presence of perisomatic GABAergic contacts, reelin+ cells also had a lower sIPSC frequency. c) Example raw traces of sIPSC measurements recorded in calb+ (left) and reelin+ (right) cells. d) An example of the immunohistochemical markers (note that colorization of fluorescent channels has been optimized for clarity between different experiments and does not represent the fluorophore used for visualization) calbindin (red), reelin (blue), PV (green), and VGAT (magenta). When PV (shown in green with reelin and calbindin on the left panel) is combined with VGAT immunohistochemistry to identify presynaptic GABAergic terminals (image shown on right demonstrating PV in green and VGAT in magenta, with locations of calb+ and reelin+ cells denoted by red squares and blue triangles, respectively), synaptic regions can be identified by colocalization, shown in white. Insets: Note that calb+ cells receive both individual PV boutons (green triangle on left panel; shown colocalizing with VGAT, white triangle, on right panel) as well as numerous VGAT positive punctae without PV (magenta arrow) (which may represent CCKBC terminals, because only two types of basket cells provide innervation to principal cells in cortical circuits, the CCKBCs and the PVBCs), while reelin+ cells have few such punctae (scale bar = 50μm). e) More PV containing punctae surround the somata of calb+ than reelin+ principal cells. e) CCKBCs, whose punctae can be identified by the marker VGLUT3, were again seen to have a strong preference for calb+ as compared to reelin+ cells in juvenile animals.

Article Snippet: For all bouton counting experiments, while electron microscopy was not performed to validate the error rate with selection of boutons, we expect that because all data were analyzed as a comparative study between calb + and reelin + cells within individual slices and, when applicable, between epileptic and sham animals in age-matched slices prepared together, the error rate of bouton identification was considered similar for both principal cell populations and between epileptic and age-matched sham control animals. table ft1 table-wrap mode="anchored" t5 caption a7 detection of: experiments antibody antibody manufacturer & catalogue number concentration reelin all primary mouse anti-reelin Millipore monoclonal AB5364 clone G10 1:2,000 secondary Alexa 647 donkey anti-mouse Jackson Immunoresearch 715-605-151 1:500 calbindin all except PV axon tracing primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary Alexa 594 donkey anti-rabbit Jackson Immunoresearch 711-585-152 1:500 calbindin PV cell axon tracing only primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary DyLight 405 donkey anti-rabbit Jackson Immunoresearch 711-475-152 1:500 PV all PV immunostaining primary goat anti-parvalbumin Swant polyclonal antibody number PVG213 1:1,000 secondary Alexa 488 donkey anti-goat Jackson Immunoresearch 705-545-147 1:500 VGLUT3 all VGLUT3 immunostaining primary guinea pig anti-VGLUT3 Chemicon polyclonal antibody, AB5421 1:10,000 secondary Alexa 488 donkey anti-guinea pig Jackson Immunoresearch 706-545-148 1:500 VGAT PV bouton counting only primary guinea pig anti-VGAT Synaptic Systems polyclonal antibody, number 131 004 1:1,000 secondary Alexa 594 donkey anti-guinea-pig Jackson Immunoresearch 706-585-148 1:500 cell fills all except PV axon tracing primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary DyLight 405 streptavidin Jackson Immunoresearch 016-470-084 1:200 cell fills PV cell axon tracing only primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary Alexa 594 streptavidin Jackson Immunoresearch 016-580-084 1:200 Open in a separate window caption a8 Antibodies and other reagents used for cell detection and associated catalogue information See methods and results for descriptions of use.

Techniques: Immunohistochemical staining, Immunohistochemistry, Marker

Journal: Hippocampus

Article Title: Target-Selectivity of Parvalbumin-Positive Interneurons in Layer II of Medial Entorhinal Cortex in Normal and Epileptic Animals

doi: 10.1002/hipo.22559

Figure Lengend Snippet: Intrinsic properties of calb + and reelin + cells in juvenile control animals, adult sham control animals, and adult epileptic animals See methods section and for descriptions of each of the measured intrinsic properties. Subscript 1 indicates the properties of the first spike in the most depolarized sweep. Properties with a Δ in front of them indicate a difference between the first spike and the subsequent 3 spikes in the most depolarized sweep. Note that because there were no differences in any of the measured parameters between epileptic and age-matched sham control animals, the right-hand column does not display the latter comparison.

Article Snippet: For all bouton counting experiments, while electron microscopy was not performed to validate the error rate with selection of boutons, we expect that because all data were analyzed as a comparative study between calb + and reelin + cells within individual slices and, when applicable, between epileptic and sham animals in age-matched slices prepared together, the error rate of bouton identification was considered similar for both principal cell populations and between epileptic and age-matched sham control animals. table ft1 table-wrap mode="anchored" t5 caption a7 detection of: experiments antibody antibody manufacturer & catalogue number concentration reelin all primary mouse anti-reelin Millipore monoclonal AB5364 clone G10 1:2,000 secondary Alexa 647 donkey anti-mouse Jackson Immunoresearch 715-605-151 1:500 calbindin all except PV axon tracing primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary Alexa 594 donkey anti-rabbit Jackson Immunoresearch 711-585-152 1:500 calbindin PV cell axon tracing only primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary DyLight 405 donkey anti-rabbit Jackson Immunoresearch 711-475-152 1:500 PV all PV immunostaining primary goat anti-parvalbumin Swant polyclonal antibody number PVG213 1:1,000 secondary Alexa 488 donkey anti-goat Jackson Immunoresearch 705-545-147 1:500 VGLUT3 all VGLUT3 immunostaining primary guinea pig anti-VGLUT3 Chemicon polyclonal antibody, AB5421 1:10,000 secondary Alexa 488 donkey anti-guinea pig Jackson Immunoresearch 706-545-148 1:500 VGAT PV bouton counting only primary guinea pig anti-VGAT Synaptic Systems polyclonal antibody, number 131 004 1:1,000 secondary Alexa 594 donkey anti-guinea-pig Jackson Immunoresearch 706-585-148 1:500 cell fills all except PV axon tracing primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary DyLight 405 streptavidin Jackson Immunoresearch 016-470-084 1:200 cell fills PV cell axon tracing only primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary Alexa 594 streptavidin Jackson Immunoresearch 016-580-084 1:200 Open in a separate window caption a8 Antibodies and other reagents used for cell detection and associated catalogue information See methods and results for descriptions of use.

Techniques:

Journal: Hippocampus

Article Title: Target-Selectivity of Parvalbumin-Positive Interneurons in Layer II of Medial Entorhinal Cortex in Normal and Epileptic Animals

doi: 10.1002/hipo.22559

Figure Lengend Snippet: Epileptic animals were generated using the low dose kainate model and monitored for spontaneous recurrent seizures using continuous video and EEG monitoring. a) An example of a spontaneous behavioral and electrographic seizure recorded in a chronically epileptic rat used for this study. b) An example of the immunohistochemical markers in tissue from epileptic animals (note that colorization of fluorescent channels has been optimized for clarity between different experiments and does not represent the fluorophore used for visualization): calbindin (red), reelin (blue), PV (green), and VGLUT3 (yellow). Left panel shows PV, calbindin, and reelin only, while right panel shows VGLUT3, calbindin, and reelin only. (scale bar = 10μm). c) In both age-matched sham controls and chronically epileptic animals, PV basket cells (PVBCs) preferentially formed perisomatic punctae around calbindin (calb+) containing compared to reelin (reelin+) containing principal cells, and d) VGLUT3 positive perisomatic punctae had a strong quantitative preference for calb+ compared to reelin+ cells, which did not differ in epileptic animals. e) Miniature inhibitory postsynaptic currents (mIPSCs) were reduced in epileptic compared to age-matched sham controls.

Article Snippet: For all bouton counting experiments, while electron microscopy was not performed to validate the error rate with selection of boutons, we expect that because all data were analyzed as a comparative study between calb + and reelin + cells within individual slices and, when applicable, between epileptic and sham animals in age-matched slices prepared together, the error rate of bouton identification was considered similar for both principal cell populations and between epileptic and age-matched sham control animals. table ft1 table-wrap mode="anchored" t5 caption a7 detection of: experiments antibody antibody manufacturer & catalogue number concentration reelin all primary mouse anti-reelin Millipore monoclonal AB5364 clone G10 1:2,000 secondary Alexa 647 donkey anti-mouse Jackson Immunoresearch 715-605-151 1:500 calbindin all except PV axon tracing primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary Alexa 594 donkey anti-rabbit Jackson Immunoresearch 711-585-152 1:500 calbindin PV cell axon tracing only primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary DyLight 405 donkey anti-rabbit Jackson Immunoresearch 711-475-152 1:500 PV all PV immunostaining primary goat anti-parvalbumin Swant polyclonal antibody number PVG213 1:1,000 secondary Alexa 488 donkey anti-goat Jackson Immunoresearch 705-545-147 1:500 VGLUT3 all VGLUT3 immunostaining primary guinea pig anti-VGLUT3 Chemicon polyclonal antibody, AB5421 1:10,000 secondary Alexa 488 donkey anti-guinea pig Jackson Immunoresearch 706-545-148 1:500 VGAT PV bouton counting only primary guinea pig anti-VGAT Synaptic Systems polyclonal antibody, number 131 004 1:1,000 secondary Alexa 594 donkey anti-guinea-pig Jackson Immunoresearch 706-585-148 1:500 cell fills all except PV axon tracing primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary DyLight 405 streptavidin Jackson Immunoresearch 016-470-084 1:200 cell fills PV cell axon tracing only primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary Alexa 594 streptavidin Jackson Immunoresearch 016-580-084 1:200 Open in a separate window caption a8 Antibodies and other reagents used for cell detection and associated catalogue information See methods and results for descriptions of use.

Techniques: Generated, Immunohistochemical staining

Journal: Hippocampus

Article Title: Target-Selectivity of Parvalbumin-Positive Interneurons in Layer II of Medial Entorhinal Cortex in Normal and Epileptic Animals

doi: 10.1002/hipo.22559

Figure Lengend Snippet: a) An example of a direct excitatory connection recorded from a calbindin (calb+) to a reelin (reelin+) containing principal cell in an epileptic animal (inset). b) Another example pair, this time between two reelin+ cells (inset). For both panels: recordings performed in a K-gluconate based solution. Upper traces show the presynaptic spike, lower traces demonstrate the response in the postsynaptic cell in ACSF (dark blue, dark red) that was abolished in AMPA/kainate and NMDA antagonists, NBQX and APV (light blue, light red).

Article Snippet: For all bouton counting experiments, while electron microscopy was not performed to validate the error rate with selection of boutons, we expect that because all data were analyzed as a comparative study between calb + and reelin + cells within individual slices and, when applicable, between epileptic and sham animals in age-matched slices prepared together, the error rate of bouton identification was considered similar for both principal cell populations and between epileptic and age-matched sham control animals. table ft1 table-wrap mode="anchored" t5 caption a7 detection of: experiments antibody antibody manufacturer & catalogue number concentration reelin all primary mouse anti-reelin Millipore monoclonal AB5364 clone G10 1:2,000 secondary Alexa 647 donkey anti-mouse Jackson Immunoresearch 715-605-151 1:500 calbindin all except PV axon tracing primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary Alexa 594 donkey anti-rabbit Jackson Immunoresearch 711-585-152 1:500 calbindin PV cell axon tracing only primary rabbit anti-calbindin Swant polyclonal antibody D-28k, number CB38 1:5,000 secondary DyLight 405 donkey anti-rabbit Jackson Immunoresearch 711-475-152 1:500 PV all PV immunostaining primary goat anti-parvalbumin Swant polyclonal antibody number PVG213 1:1,000 secondary Alexa 488 donkey anti-goat Jackson Immunoresearch 705-545-147 1:500 VGLUT3 all VGLUT3 immunostaining primary guinea pig anti-VGLUT3 Chemicon polyclonal antibody, AB5421 1:10,000 secondary Alexa 488 donkey anti-guinea pig Jackson Immunoresearch 706-545-148 1:500 VGAT PV bouton counting only primary guinea pig anti-VGAT Synaptic Systems polyclonal antibody, number 131 004 1:1,000 secondary Alexa 594 donkey anti-guinea-pig Jackson Immunoresearch 706-585-148 1:500 cell fills all except PV axon tracing primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary DyLight 405 streptavidin Jackson Immunoresearch 016-470-084 1:200 cell fills PV cell axon tracing only primary biocytin-filled cell Sigma-Aldrich B4261 8mM secondary Alexa 594 streptavidin Jackson Immunoresearch 016-580-084 1:200 Open in a separate window caption a8 Antibodies and other reagents used for cell detection and associated catalogue information See methods and results for descriptions of use.

Techniques: