Journal: bioRxiv

Article Title: A neural circuit targeting technique for investigating functional input-output organization in the nervous system

doi: 10.1101/2025.04.17.649280

Figure Lengend Snippet: a-c, Description of conventional anatomical connectivity-defined targeting of neuronal populations by anterograde transneuronal or retrograde viruses in a simplified input-output circuit. a, Schematic diagram of neural circuitry showing information flow through a target region receiving afferent inputs from two regions and sending efferent outputs to two target regions. b, Conventional output-based circuit-based cell type targeting with retrograde virus. The targeted cells project to a given region and receive input from multiple regions. c, Input-based circuit element targeting using transneuronal anterograde virus. These cells receive inputs from a specific region and project to multiple regions. d, Identification of the dual-transneuronal input-output circuit from the cuneiform nucleus (CnF) through the lateral and basal nuclei of the amygdala (LA/B) to the central nucleus of the amygdala (CeA). Schematic (left) of viral combinations to co-label LA/B neurons receiving input from CnF and sending output to CeA. Representative image of neurons (arrowheads) in LA co-expressing CnF-input (tdTomato) and CeA-output (eGFP) labels (replicated in N = 3 animals). e, Anterograde Cre and retrograde Cre-dependent fluorophore experiment. Schematic (left) of viral combinations and example image (right) showing labeling in LA/B and CeA. f-h, Reversing the injection site of the two splitCre-PINCER components (NCre and CCre) can capture the CnF-input/CeA-output neurons in LA/B. f, Schematic showing of reversed viral combinations. g, Reagents and pAAV plasmid sequence design used in the target site reversed splitCre-PINCER approaches. Open and filled triangles, incompatible loxP sites. h, Representative image of the CnF-input/CeA-output neurons (rAAV5-CAG-cDIO-tdTomato+) labeled by the reversed splitCre-PINCER approach (replicated in N = 3 animals). Scale bars, d, 100μm, e,h, 500μm. e,h Bregma distance in anterior-posterior direction.

Article Snippet: rAAV1-hSyn-Cre (pENN.AAV.hSyn.Cre.WPRE.hGH, a gift from James M. Wilson, Addgene viral prep #10553-AAV1), rAAV5-CAG-tdTomato (pAAV-CAG-tdTomato, a gift from Edward Boyden, Addgene viral prep #59462-AAV5), rAAV5-CAG-eGFP (pAAV-CAG-GFP, a gift from Edward Boyden, Addgene viral prep #37825-AAV5), rAAV5-CAG-cDIO-tdTomato (pAAV-FLEX-tdTomato, a gift from Edwad Boyden, Addgene viral prep #28306-AAV5), rAAV5-Syn-cDIO-GCaMP6s (pAAV.Syn.Flex.GCaMP6s.WPRE.SV40, a gift from Douglas Kim and GENIE Project, Addgene viral prep #100845-AAV5) , rAAV5-CAG-cDIO-ArchT-tdTomato (pAAV-FLEX-ArchT-tdTomato, a gift from Edward Boyden, Addgene viral prep #28305-AAV5) , retroAAV2-CAG-GFP (pAAV-CAG-GFP, a gift from Edward Boyden, Addgene viral prep #37825-AAVrg) and retroAAV2-CAG-tdTomato (pAAV-CAG-tdTomato, a gift from Edward Boyden, Addegene viral perp #59462-AAVrg) were purchased from Addgene.

Techniques: Virus, Expressing, Labeling, Injection, Plasmid Preparation, Sequencing

Journal: bioRxiv

Article Title: A neural circuit targeting technique for investigating functional input-output organization in the nervous system

doi: 10.1101/2025.04.17.649280

Figure Lengend Snippet: a-b , The concept, logic, and reagents for ‘PINCER’ method. a , Using transneuronal anterograde and retrograde viruses to express recombinases, PINCER captures a neural population based on both their afferent inputs and efferent outputs. FP: fluorescent protein. b, Schematic ( top, left ) showing the splitCre-PINCER approach in the lateral and basal nuclei of the amygdala (LA/B) for labeling cells based on the inputs they receive from the cuneiform nucleus (CnF) and the outputs they send to the central nucleus of the amygdala (CeA). Reagents ( bottom ) used and pAAV plasmid design for the splitCre-PINCER approach. Open and filled triangles, incompatible loxP sites. Representative image ( top, right ) showing successful implementation of PINCER to label the CnF-input and CeA-output neurons in LA/B (CnF-input/CeA-output neurons, rAAV5-cDIO-CAG-tdTomato+, magenta cells). Area of green labeled cells from CeA co-injection of rAAV5-CAG-eGFP+ without eGFP expression in LA/B demonstrates no leakage of retrograde virus from CeA into LA/B. c-e , Co-expression analysis of glutamatergic ( vGluT ) and GABAergic ( vGAT ) neuronal markers in the CnF-input alone, CeA-output alone and CnF-input/CeA-output neurons in LA/B. c, Schematic of viral combinations to label the CnF-input, CeA-output, and CnF-input/CeA-output neurons to co-label them with vGluT and vGAT mRNAs with the hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) in LA/B. d, Representative images ( left ) of the CnF-input alone ( top ), CeA-output alone ( middle ), and CnF-input/CeA-output ( bottom ) neurons expressing vGluT in LA/B. Arrowheads and arrows denote anatomical connectivity defined neurons which are vGluT+ or vGlut- , respectively. Pie charts ( right ) showing the percentage (mean±SEM) of the CnF-input alone neurons ( top , N = 4 animals, total of n = 58 CnF-input neurons analyzed), CeA-output alone neurons (middle, N = 3 animals, total of n = 552 CeA-output neurons analyzed), or CnF-input/CeA-output neurons (bottom, N = 4 animals, total of n = 60 CnF-input/CeA-output neurons analyzed) co-labelled with vGluT in LA/B. e, Representative images ( left ) of the CnF-input alone ( top ), CeA-output alone ( middle ) and CnF-input/CeA-output ( bottom ) labeled neurons and neurons expressing vGAT in LA/B. Arrowheads and arrows denote anatomical connectivity defined neurons which are vGAT+ or vGAT- , respectively. Pie charts ( right ) showing the percentage (mean±SEM) of the CnF-input alone neurons ( top , N = 4 animals, total of n = 58 CnF-input neurons analyzed), CeA-output alone neurons ( middle , N = 3 animals, total of n = 552 CeA-output neurons analyzed) or CnF-input/CeA-output neurons ( bottom , N = 4 animals, total of n = 60 CnF-input/CeA-output neurons analyzed) co-labelled with vGAT in LA/B. Scale bars, b, 500μm, d,e, 30μm. b, Bregma distance in anterior-posterior direction.

Article Snippet: rAAV1-hSyn-Cre (pENN.AAV.hSyn.Cre.WPRE.hGH, a gift from James M. Wilson, Addgene viral prep #10553-AAV1), rAAV5-CAG-tdTomato (pAAV-CAG-tdTomato, a gift from Edward Boyden, Addgene viral prep #59462-AAV5), rAAV5-CAG-eGFP (pAAV-CAG-GFP, a gift from Edward Boyden, Addgene viral prep #37825-AAV5), rAAV5-CAG-cDIO-tdTomato (pAAV-FLEX-tdTomato, a gift from Edwad Boyden, Addgene viral prep #28306-AAV5), rAAV5-Syn-cDIO-GCaMP6s (pAAV.Syn.Flex.GCaMP6s.WPRE.SV40, a gift from Douglas Kim and GENIE Project, Addgene viral prep #100845-AAV5) , rAAV5-CAG-cDIO-ArchT-tdTomato (pAAV-FLEX-ArchT-tdTomato, a gift from Edward Boyden, Addgene viral prep #28305-AAV5) , retroAAV2-CAG-GFP (pAAV-CAG-GFP, a gift from Edward Boyden, Addgene viral prep #37825-AAVrg) and retroAAV2-CAG-tdTomato (pAAV-CAG-tdTomato, a gift from Edward Boyden, Addegene viral perp #59462-AAVrg) were purchased from Addgene.

Techniques: Labeling, Plasmid Preparation, Injection, Expressing, Virus, Hybridization, Fluorescence, In Situ Hybridization

Journal: Molecular Psychiatry

Article Title: Dysfunctional serotonergic neuron-astrocyte signaling in depressive-like states

doi: 10.1038/s41380-023-02269-8

Figure Lengend Snippet: a Top, Scheme of corticosterone (Cort) treatment protocol for 28 days followed by behavioral testing. Decreased doses of Cort were applied at different time points. Behavioral testing schedule. OF, EPM, OIP, NOR, SR and FST were conducted sequentially from 3 days (OF) to 9 days after treatment (FST). Bottom left, body weights for naïve (in black, as in the rest of the figure) and Cort-mice (in blue, as in the rest of the figure) were monitored during the entire Cort treatment. Naïve mice were exposed to water as control. Note the reduced gain of weight for Cort-mice ( n = 26 for naïve; n = 29 for Cort). One Way ANOVA, Holm-Sidak method. P = 0.002, P < 0.001. Data are presented as mean ± s.e.m. Bottom right, cumulative duration of immobility during force swimming test (FST) in naïve ( n = 26) and Cort mice ( n = 29), showing an increased immobility rates compare to their naïve littermates. One Way ANOVA, Dunn’s test. P < 0.001. Data are shown as median ± range (min and max values). b Top, schematic representation of mPFC area targeted with AAV5-GFAP-GCaMP6f viral injection and optical fiber implantation in naïve and Cort-mice. Confocal image showing the selective viral expression (green) in astrocytes (S100β, red) in mPFC. Scale bar: 500 μm. Bottom left, representative confocal images showing GCaMP6f expression and astrocytic (S100β) and neuronal markers (NeuN) labeling. Scale bar: 20 μm. Bottom right, colocalization analysis of GCaMP6f positive cells and S100β and NeuN labeling (162 GCaMP6f positive cells from 3 slices, 3 mice; One Way ANOVA, Tukey test, P < 0,001). c Representative in vivo astrocytic Ca 2+ activity traces from fiber photometry recordings and speed in naïve (top) and Cort-mice (bottom) during open field explorations. Dots denote the peak of astrocyte Ca 2+ events. d Box and whisker (BW) plots analysis of Ca 2+ dynamics in both groups (naïve= 7, Cort= 6). One Way ANOVA, Dunn ´s Method, P > 0.05, P < 0.05, <0.01. e Representative spatial heatmaps for naïve and Cort-mice during a social recognition task. Color code denotes accumulated time. f Behavioral analysis of social preference for naïve and Cort-mice both implanted with the fiber photometry cannula (FP; naïve = 14, Cort = 15), and mice without surgical manipulations (Control, naïve = 14, Cort = 8), showing similar performance. Note that cannula implantation did not alter social preference. Cort-mice did not display preference for social interaction. Two Way ANOVA, Holm-Sidak method. P > 0.05. Y-axis represents the time spent exploring either the unfamiliar mouse (orange) or object (gray). g Representative astrocytic Ca 2+ activity recordings from fiber photometry in naïve and Cort-mice during a social recognition task. Shaded areas indicate interaction bouts with unfamiliar mouse (orange) or object (gray), and dots indicate astrocyte Ca 2+ events. h Z-score of astrocytic Ca 2+ response during mouse (continuous line) and object (dashed line) interaction bouts for naïve ( n = 7) and Cort-mice ( n = 6). Events were aligned to the exploration onset. Reduced amplitude of Ca 2+ signals upon mouse interaction was found in Cort-mice (n Cort =26, n naïve = 34 Ca 2+ events). One Way ANOVA, Dunn ´s Method, P = 0.031. The center line in BW plots indicates the median, the top and bottom edges indicate the 25th and 75th percentiles, respectively, and the whiskers extend to the maximum and minimum data points. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following viral vectors were used: AAV5-gfaABC1D-cyto-GCaMP6f (Addgene 52925; viral titer 1.3 × 10 13 GC/ml), AAV5-GFAP-hM3Dq-mCherry (Addgene 50478; viral titer 2 × 10 13 GC/ml), AAV8-GFAP104-mCherry (UNC Vector Core; viral titer 2.7 × 10 12 GC/ml), AAV9-hSyn-ChrimsonR-tdTom (UNC Vector Core; viral titer 4.1 × 10 12 GC/ml), AAV5-GFAP-mcherry-cre (UNC Vector Core; viral titer 4.3 × 10 12 GC/ml), AAV5-CAG-flex-iSeroSnFR (Addgene 128486; viral titer 5 x + 10 12 GC/ml, Unitat de Vectors Virals, UAB), AAV5-GFAP-eGFP-WPRE-hGH (Addgene 105549; viral titer 1.3 × 10 13 GC/ml), AAV9-CaMKIIa-eYFP (Penn Vector Core, viral titer 2.55 × 10 12 GC/ml).

Techniques: Injection, Expressing, Labeling, In Vivo, Activity Assay, Whisker Assay

Journal: Molecular Psychiatry

Article Title: Dysfunctional serotonergic neuron-astrocyte signaling in depressive-like states

doi: 10.1038/s41380-023-02269-8

Figure Lengend Snippet: a Left. Sagittal and coronal scheme of viral injection in the mPFC (green shaded area) to target astrocytes using AAV5-GFAP-GCaMP6f virus. Right . Squeme of spontaneous and 5-HT-evoked calcium events in mPFC astrocytes. b Heatmaps of spontaneous ROIs activity and average population activity in astrocytes of naïve (black, n = 117 ROIs, n = 3 mice) and Cort-mice (blue, n = 190 ROIs; n = 2 mice). Data are presented as mean ± s.e.m. Color code denotes fluorescence changes. c Box and whisker (BW) plots representing the dynamics of Ca 2+ astrocytic events in naïve and Cort mice. Cort-mice showed an increased frequency of Ca 2+ events ( P = 0.006) but decreased amplitude, area and duration of events. One Way ANOVA, Dunn’s Method, P < 0.001. d Heatmaps of 5-HT evoked ROIs activity and average population activity in astrocytes of naïve ( n = 200, n = 3 mice) and Cort-mice ( n = 350, n = 2 mice). Red triangle and bar denote 5-HT puff application (1 mM, 10 s, 1 bar). e BW plots representing the dynamics of Ca 2+ astrocytic events in response to 5-HT of naïve and Cort-mice. Reduced amplitude and area of Ca 2+ events were found in Cort-mice (black bars vs dark blue bars). One Way ANOVA, Dunn’s Method, P < 0.001. f Heatmaps of 5-HT evoked ROIs activity and average population activity in astrocytes of naïve mice, in basal conditions (left)and in presence of ketanserin (right, n = 75, n = 2 mice). g Representative traces of Ca 2+ astrocytic signals from naïve and Cort-mice evoked by local 5-HT puff (red triangle) in control conditions (left, black) and after bath application of ketanserin (right, purple). h , i BW plots representing changes in amplitude and frequency of Ca 2+ events induced local 5-HT stimulation before (black) and after bath application of 5-HT2R family antagonists (ketanserin, purple; RS127445, orange, n = 70, n = 2 mice; RS102221, green, n = 69, n = 2 mice) in naïve mice ( h ), and Cort-mice (ketanserin, n = 47, n = 2 mice; RS127445, n = 70, n = 2 mice; RS102221, n = 51, n = 2 mice) ( i ). Ca 2+ event amplitude and frequency induced by 5-HT was normalized to spontaneous activity before 5-HT stimulation (black bars). These parameters were normalized to the Ca 2+ signals evoked by 5-HT before 5-HT2R antagonists family blockade. Note the reduced activity under the influence of each antagonist. P < 0.001. One Way ANOVA, Dunn’s method. j Representative traces of slow inward currents (SICs) before and after local 5-HT application (1 mM, 10 s, 1 bar) in naïve and Cort-mice. Red asterisk indicates the presence of SICs. k Scatter plot of SICs recorded before (5 min) and after 5-HT (5 min) showing a significant increase in frequency after 5-HT stimulation in naïve mice ( n = 16 cells, n = 2 mice; Tukey Test, P < 0.001), without further changes in current amplitude for both naïve and Cort-mice ( n = 19 cells, n = 3 mice). One Way ANOVA, Dunn’s Method, P = 0.758 for naïve, P = 0.982 for Cort. Note the enhanced SICs frequency in resting conditions for Cort-mice, which were insensitive to further increase by 5-HT stimulation. One Way ANOVA, Dunn’s Method, P = 0.033. Data shown as median ± range (min and max values). The center line in BW plots indicates the median, the top and bottom edges indicate the 25th and 75th percentiles, respectively, and the whiskers extend to the maximum and minimum data points. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following viral vectors were used: AAV5-gfaABC1D-cyto-GCaMP6f (Addgene 52925; viral titer 1.3 × 10 13 GC/ml), AAV5-GFAP-hM3Dq-mCherry (Addgene 50478; viral titer 2 × 10 13 GC/ml), AAV8-GFAP104-mCherry (UNC Vector Core; viral titer 2.7 × 10 12 GC/ml), AAV9-hSyn-ChrimsonR-tdTom (UNC Vector Core; viral titer 4.1 × 10 12 GC/ml), AAV5-GFAP-mcherry-cre (UNC Vector Core; viral titer 4.3 × 10 12 GC/ml), AAV5-CAG-flex-iSeroSnFR (Addgene 128486; viral titer 5 x + 10 12 GC/ml, Unitat de Vectors Virals, UAB), AAV5-GFAP-eGFP-WPRE-hGH (Addgene 105549; viral titer 1.3 × 10 13 GC/ml), AAV9-CaMKIIa-eYFP (Penn Vector Core, viral titer 2.55 × 10 12 GC/ml).

Techniques: Injection, Virus, Activity Assay, Fluorescence, Whisker Assay

Journal: Molecular Psychiatry

Article Title: Dysfunctional serotonergic neuron-astrocyte signaling in depressive-like states

doi: 10.1038/s41380-023-02269-8

Figure Lengend Snippet: a Confocal imaging of immunohistochemistry-confirmed hM3Dq-mCherry expression (red) in astrocytes (S100-β, green). Scale bar, 500 μm (up), 50 μm (down). b Heatmaps of CNO-hM3D(Gq) evoked ROIs activity and average population activity in astrocytes of naïve ( n = 164, n = 3 mice) and Cort-mice ( n = 200, n = 2 mice). Red triangle and bar denote CNO puff application (1 mM, 2 s, 1 bar). Data are presented as mean ± s.e.m. Color code denotes fluorescence change. c Box and whisker (BW) plots representing fluctuations of Ca 2+ astrocytic events induced by CNO in naïve and Cort-mice. Both mice showed an increased in normalized CNO-evoked amplitude, Paired t test, P < 0.001. d Scheme and representative activity traces in EPM for naïve, Cort-mice and Cort-GFAP-hM3Dq mice stimulated with CNO (3 mg/kg, i.p.). e EPM exploration index was enhanced in Cort-GFAP-hM3Dq mice vs Cort-mice, showing more entries into the open arms. One Way ANOVA, Dunn’s method, P = 0.003. Data are presented as median ± range for the entire figure. f FST immobility time was rescued in Cort-GFAP-hM3Dq mice (One Way ANOVA, Tukey test, P < 0.001), reaching similar values to naïve mice (One Way ANOVA, P = 0.413). g OIP discrimination index reduced in Cort-mice ( P < 0.001) was increased after CNO administration. One Way ANOVA, Tukey test, P < 0.001. h OIP test scheme and representative spatial heatmaps of naïve and Cort-mice performing the task. Color code denotes accumulated time. i NOR discrimination index reduced in Cort-mice (One Way ANOVA, Tukey test, P = 0.001) was rescued after CNO hM3Dq-astrocyte stimulation (One Way ANOVA, Holm-Sidak method, P = 0.021). j Representative spatial heatmaps of naïve and Cort-mice during SR task. Color code denotes accumulated time. k Exploration time analyzed for naïve, Cort-mice and Cort-GFAP-hM3Dq mice during SR test showing that reduced levels of social interaction found in Cort-mice (One Way ANOVA, Holm-Sidak method, P = 0.004) were reverted by CNO administration (One Way ANOVA, Dunn´s method, P = 0.030). l EPM exploration index in naïve GFAP-hM3Dq mice was reduced compared with control GFAP-mcherry naïve mice after CNO administration (3 mg/kg, i.p.). One Way ANOVA, Holm-Sidak, P = 0.007. m FST immobility time showed enhanced values in GFAP-hM3Dq mice. One Way ANOVA, Tukey Test, P = 0.002. n , o , p NOR discrimination index, OIP discrimination index and SR exploration time were reduced in GFAP-hM3Dq mice. One Way ANOVA, Dunn’s Method, P < 0.001 for NOR; Tukey Test, P = 0.011 for OIP; Tukey Test, P = 0,015 for SR. q Scheme of CNO and AIDA (5 mg/kg) i.p. administration in Cort-GFAP-hM3Dq mice. 1 and 2 denotes sequential i.p. injections. r FST immobility time showing increased values for Cort-GFAP-hM3Dq+AIDA mice (gray) compared with Cort-GFAP-hM3Dq mice (red, shown as median ± range as reference). One Way ANOVA, Tukey test P = 0,008. s , t , u NOR discrimination index, OIP discrimination index and SR exploration time were reduced by previous administration of AIDA in Cort-GFAP-hM3Dq mice. One Way ANOVA, Dunn’s Method, P = 0,020 for NOR; Tukey test, P < =0.001 for OIP; Dunn’s Method, P = 0,015 for SR. The center line in plots indicates the median, the top and bottom edges indicate the 25th and 75th percentiles, respectively, and the whiskers extend to the maximum and minimum data points. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The following viral vectors were used: AAV5-gfaABC1D-cyto-GCaMP6f (Addgene 52925; viral titer 1.3 × 10 13 GC/ml), AAV5-GFAP-hM3Dq-mCherry (Addgene 50478; viral titer 2 × 10 13 GC/ml), AAV8-GFAP104-mCherry (UNC Vector Core; viral titer 2.7 × 10 12 GC/ml), AAV9-hSyn-ChrimsonR-tdTom (UNC Vector Core; viral titer 4.1 × 10 12 GC/ml), AAV5-GFAP-mcherry-cre (UNC Vector Core; viral titer 4.3 × 10 12 GC/ml), AAV5-CAG-flex-iSeroSnFR (Addgene 128486; viral titer 5 x + 10 12 GC/ml, Unitat de Vectors Virals, UAB), AAV5-GFAP-eGFP-WPRE-hGH (Addgene 105549; viral titer 1.3 × 10 13 GC/ml), AAV9-CaMKIIa-eYFP (Penn Vector Core, viral titer 2.55 × 10 12 GC/ml).

Techniques: Imaging, Immunohistochemistry, Expressing, Activity Assay, Fluorescence, Whisker Assay