Journal: bioRxiv

Article Title: Magnetoelectric Nanodiscs Enable Wireless Transgene-Free Neuromodulation

doi: 10.1101/2023.12.24.573272

Figure Lengend Snippet: Relative GCaMP6s fluorescence change (ΔF/F 0 ) in hippocampal neurons decorated with MENDs before (left) and after (right) magnetic field application. Scale bars: 150 µm. b, Individual (top) and average (bottom) of the relative GCaMP6s fluorescence change (ΔF/F 0 ) of the hippocampal neurons in response to 10 mT AMF with 1 kHz frequency and 220 mT OFM. c, Individual and d, average traces of GCaMP6s ΔF/F 0 in 300 hippocampal neurons decorated with MENDs in response to 10 mT AMF with frequencies 100, 150, 250, 500, and 1000 Hz (OMF magnitude 220 mT). The grey and magenta dashed lines indicate the beginning and end of MF stimulation, respectively. e, Individual cell (top) and mean (bottom) GCaMP6s fluorescence changes in 300 neurons in response to 2 s MF epochs applied at varying intervals (OMF 220 mT; AMF 150 Hz, 10 mT). f, Number of GCaMP6s fluorescence peaks as a function of stimulation epoch length for rest intervals of 10, 30, 60, 90, and 120 s (OMF 220 mT; AMF 150 Hz, 10 mT). g, Individual cell (top) and mean (bottom) GCaMP6s fluorescence changes in response to 2 s MF epochs at 40 s intervals for 0.75 µg mm −2 MEND density. h, i, Fluorescent image of a Live-Dead assay in neurons before and after 3 cycles of MF at h, 1 µg mm −2 and i, 0.75 µg mm −2 MEND densities. j, The change in live cell ratio (normalized to the total number of the cells counted with Hoest staining) following 3 cycles of MF for neurons decorated with different MEND densities. Statistical significance was tested via one-way ANOVA and Tukey’s multiple comparison tests (n = 5 plates per condition, P=3.79×10 −7 for 1 µg mm −2 ; P=0.79 for 0.794 µg mm −2 ; P=0.998 for 0.75 µg mm −2 ; ****P≤0.0001, n.s. P>0.05).

Article Snippet: 4 days following seeding, the neurons were transduced with 1 μL of an adeno-associated virus serotype 9 (AAV9) carrying a fluorescent calcium ion indicator GCaMP6s under a pan-neuronal human synapsin (hSyn) promoter (AAV9-hSyn::GCaMP6s, Addgene viral prep #100843-AAV9, >1×10 13 IU/μL).

Techniques: Fluorescence, Live Dead Assay, Staining, Comparison

Journal: bioRxiv

Article Title: Magnetoelectric Nanodiscs Enable Wireless Transgene-Free Neuromodulation

doi: 10.1101/2023.12.24.573272

Figure Lengend Snippet: Illustration of stimulation mechanism combining cable model and repeated excitation of neural activity, where d is spacing between MEND particles, a is cell radius, Δ V is the change in membrane potential per half-period of an AMF, and V 0 is the voltage generated by a single MEND. b, Calculated membrane potential change Δ V for every half-period of an AMF, as a function of V 0 and d . c, Simulated membrane potential V ( t ) after AMF onset to activate MENDs with V 0 = 0.03 mV for varying a values. The threshold potential for action potential firing, –55 mV, is indicated with the dashed line. d, Simulated membrane potential at a time t = 2 s after onset of magnetic field as a function of d , for varying V 0 values. V 0 =0.03 mV is the measured value for MEND particles in this study, highlighted in red. e, Time to reach threshold membrane potential (–55 mV) from the resting potential (–75 mV) as a function of AMF frequency ƒ AMF for varying V 0 values. f, ( i-iii ) SEM images showing MENDs decorating cultured hippocampal neurons. (ii) A higher magnification image of the area marked by a box in panel (i). (iii) MENDs on the neuron surface shaded in blue as identified by the presence of iron, titanium, and barium in energy dispersive X-ray spectroscopy. Scale bars: 20 µm (i), 5 µm (ii), 100 nm (iii). g, h, GCaMP6s fluorescence changes in neurons decorated with MEND following 2 s stimulations (OMF 220 mT; AMF 150 Hz, 10 mT, marked by vertical grey bars) in the presence of g, tetrodotoxin (TTX, 1 µM, green) or h, a cocktail of (2R)-amino-5-phosphonovaleric acid (AP5, 100 µM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM) (blue).

Article Snippet: 4 days following seeding, the neurons were transduced with 1 μL of an adeno-associated virus serotype 9 (AAV9) carrying a fluorescent calcium ion indicator GCaMP6s under a pan-neuronal human synapsin (hSyn) promoter (AAV9-hSyn::GCaMP6s, Addgene viral prep #100843-AAV9, >1×10 13 IU/μL).

Techniques: Activity Assay, Membrane, Generated, Cell Culture, Spectroscopy, Fluorescence

Journal: bioRxiv

Article Title: Magnetoelectric Nanodiscs Enable Wireless Transgene-Free Neuromodulation

doi: 10.1101/2023.12.24.573272

Figure Lengend Snippet: Schematic of MEND-mediated neuromodulation. MEND particles were injected into the mouse ventral tegmental area (VTA), and the mice were placed inside a permanent magnet field providing OMF of 220 mT and a surrounding solenoid providing AMF with an amplitude 10 mT and a frequency 150 Hz. b, Confocal images of c-Fos-expressing neurons among DAPI-marked cells in the VTA. Top left: MENDs (1.5 mg/ml) with (+) magnetic stimulation (ON); top right: MEND particles (1.5 mg/ml) without (–) magnetic stimulation (OFF); bottom left: control MNDs (1.5 mg/ml) + magnetic stimulation; bottom right: MENDs (0.5 mg/ml) + magnetic stimulation. Scale bars are 25 µm. c, Quantification of c-Fos positive neurons for the conditions shown in (B) as well as the subjects injected with PBS and exposed to magnetic field. d, Confocal images and e, quantification of c-Fos positive neurons in the medial prefrontal cortex (mPFC) for the same conditions shown in (B) and (C). f, Confocal images and g, quantification of c-Fos positive neurons in the nucleus accumbens (NAc) for the same conditions shown in b and c . d, f, Scale bars are 100 µm. c, e, g, Statistical significance was tested via one-way ANOVA and Tukey’s multiple comparison tests (n = 6 mice per condition, VTA F 3,20 =93.2 P=1.21×10 −12 ; mPFC F 3,20 =60.81 P=1.21×10 −11 ; VTA F 3,20 =62.03 P=1.29×10 −12 ; ****P≤0.0001). h, Representative confocal images showing expression of GCaMP6s in the VTA. Scale bar: 50 µm. i Fiber photometry recordings of relative GCaMP6s fluorescence change (ΔF/F 0 ) in the VTA of anaesthetized mice injected with MENDs in the same region. (Top) Individual trial ΔF/F 0 . (Bottom) Average ΔF/F 0 across trials shown above. Solid line represents mean and shaded areas mark s.e.m. (n=4 mice, 10-15 trials per mouse). The grey square box indicates magnetic field epochs (2s, OMF 220 mT, AMF 10 mT, 150 Hz). j Schematic of the place preference behavioral apparatus (top) and experimental timeline (bottom). k, Time spent in the stimulation chamber out of a total test time of 600 s, for pre-(Day 1, open markers) and post-(Day 5, solid markers) learning. Paired t-test was performed for MEND (n=11) and MND (n=7) groups, and Wilcoxon signed-rank test was performed for PBS (n=7) group because the data did not follow normal distribution. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, n.s. P>0.05). l, The change in time spent in stimulation chamber between Day 1 and Day 5. (One-way ANOVA with Tukey’s post-hoc comparison test, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, n.s. P>0.05.)

Article Snippet: 4 days following seeding, the neurons were transduced with 1 μL of an adeno-associated virus serotype 9 (AAV9) carrying a fluorescent calcium ion indicator GCaMP6s under a pan-neuronal human synapsin (hSyn) promoter (AAV9-hSyn::GCaMP6s, Addgene viral prep #100843-AAV9, >1×10 13 IU/μL).

Techniques: Injection, Expressing, Control, Comparison, Fluorescence

Journal: bioRxiv

Article Title: Neural responses to social cues in the accessory olfactory bulb are altered by context and experience

doi: 10.1101/2023.11.28.569126

Figure Lengend Snippet: Transgenic and viral strategy to record from AOB projection neurons during social interactions. (A) The AOB receives input directly from the VNO and makes, largely reciprocal, connections with subcortical brain regions devoted to social behavior (red arrows; BNST: bed nucleus of the stria terminalis; ACo: cortical amygdaloid nucleus; MeA: medial amygdala; PMCo: posteromedial cortical amygdala). (Inset) Mitral and tufted neurons are the projection neurons from AOB to the social behavior network (SBN; Glom: glomerular layer; PG: periglomerular cells; M/T: mitral and tufted cells; GC: granule cells). (B) Fiberphometry recordings from AOB mitral and tufted cells rely on conditional AAV-driven expression of GCaMP in a Tbet:Cre mouse paired with a fiberoptic probe implanted in the AOB . (C) Histology showing accurate GCaMP expression (cyan) in M/T cells.

Article Snippet: We injected a conditional GCaMP expressing AAV virus (AAV9:FLEX:GCaMP6s; Addgene Plasmid #:100845; ) in the AOB (Bregma 3.5, Lateral 1.0, Depth 1.0) of Tbet-cre mice to target M/T cells in the AOB.

Techniques: Transgenic Assay, Expressing

Journal: bioRxiv

Article Title: Neural responses to social cues in the accessory olfactory bulb are altered by context and experience

doi: 10.1101/2023.11.28.569126

Figure Lengend Snippet: Strategy for automating social interactions during fiberphotometric recording. (A) Trace of mouse position of over 30 seconds (color: intensity of AOB GCaMP signal; M: male; F: female; P: predator; C: control). (B) Social stimuli were presented once every 2 minutes for a 1-minute duration and behavior states were manually scored (exploratory behavior, social interaction, approach, and investigation). (C) Top: density plots indicating the location of an animal during the time a stimulus is available (Left; all time; center left: male stimulus; center: female; center right: predator; right; empty cup). Bottom: the average time (percent of total) spent investigating a stimulus when the stimulus is available (open) or not available (closed; asterisks indicate p<0.0001, paired t-test). (D) Percentage of stimulus presentations that resulted in a successful investigation by the experimental animal (error bars: SEM). (E) Fraction of stimulus presentation events that resulted in a successful investigation event for 7 successive presentations of each stimulus (3 different randomly interleaved stimuli; 21 total) when there was not a predator (left) or was a predator (right) in the stimulus set (red: female; blue: male; green: predator; grey: empty cup; shaded regions: SEM).

Article Snippet: We injected a conditional GCaMP expressing AAV virus (AAV9:FLEX:GCaMP6s; Addgene Plasmid #:100845; ) in the AOB (Bregma 3.5, Lateral 1.0, Depth 1.0) of Tbet-cre mice to target M/T cells in the AOB.

Techniques: Control

Journal: bioRxiv

Article Title: Neural responses to social cues in the accessory olfactory bulb are altered by context and experience

doi: 10.1101/2023.11.28.569126

Figure Lengend Snippet: Example of sensory responses of AOB neurons to social and predator stimuli in an adult female mouse. (A) Video frames surrounding a social interaction, with 0 seconds (center) aligned to the onset of stimulus investigation (Blue shading indicates the availability of the male stimulus). The predator (P) and female (F) stimuli were closed during the duration of the shown frames (bracket indicates the investigation event in panel B highlighted in panel A). (B) GCaMP signal (black line; z-score normalized) during seven randomized presentations of three stimuli (male, female, predator; order indicated by the colored boxes). Vertical dashed lines indicate the start of all investigations of the male stimulus. (C) Average responses (from panel B) aligned to the start of investigation of male, female, and predator stimuli (shading: SEM).

Article Snippet: We injected a conditional GCaMP expressing AAV virus (AAV9:FLEX:GCaMP6s; Addgene Plasmid #:100845; ) in the AOB (Bregma 3.5, Lateral 1.0, Depth 1.0) of Tbet-cre mice to target M/T cells in the AOB.

Techniques:

Journal: bioRxiv

Article Title: Neural responses to social cues in the accessory olfactory bulb are altered by context and experience

doi: 10.1101/2023.11.28.569126

Figure Lengend Snippet: Average GCaMP response across stimulus presentations. (A) Average GCaMP signal of female (red), male (blue), predator (green) and control (grey) stimuli relative to the first investigation after a stimulus became available (shading: SEM; N = 16: 7 males, 9 females). (B). Average z-score during the 20 seconds following sensory investigation for female, male, predator, and control stimuli. Error bars: SEM; asterisks: p-value<0.000001.

Article Snippet: We injected a conditional GCaMP expressing AAV virus (AAV9:FLEX:GCaMP6s; Addgene Plasmid #:100845; ) in the AOB (Bregma 3.5, Lateral 1.0, Depth 1.0) of Tbet-cre mice to target M/T cells in the AOB.

Techniques: Control

Journal: bioRxiv

Article Title: Neural responses to social cues in the accessory olfactory bulb are altered by context and experience

doi: 10.1101/2023.11.28.569126

Figure Lengend Snippet: AOB response to social stimuli depends on context. (A) Left, average GCaMP signal (z-score normalized) across presentations of female stimuli during experiments that included predator stimuli (red) or did not include predator stimuli (grey). Right, mean GCaMP response to female stimuli during predator and no predator experiments (t-test comparison p value = 0.011, error bars: SEM). (B) Pseudocolored plot of the average response to female stimuli for each of 7 presentations during the predator condition (top) or no predator condition (bottom). (C) Effect of repeated presentations of female stimuli on the average GCaMP response (0-20 seconds) for predator (red) and no predator (gray; error bars: SEM). (D) Left, average GCaMP signal (z-score normalized) across presentations of male stimuli during experiments that included predator stimuli (blue) or did not include predator stimuli (grey). Right, mean GCaMP response to male stimuli during predator and no predator experiments (t-test comparison p value = 0.018; error bars: SEM). (E) Pseudocolored plot of the average response to male stimuli for each of 7 presentations during the predator condition (top) or no predator condition (bottom). (F) Effect of repeated presentations of male stimuli on the average GCaMP response (0-20 seconds) for predator (blue) and no predator (gray; error bars: SEM).

Article Snippet: We injected a conditional GCaMP expressing AAV virus (AAV9:FLEX:GCaMP6s; Addgene Plasmid #:100845; ) in the AOB (Bregma 3.5, Lateral 1.0, Depth 1.0) of Tbet-cre mice to target M/T cells in the AOB.

Techniques: Comparison

Journal: bioRxiv

Article Title: Neural responses to social cues in the accessory olfactory bulb are altered by context and experience

doi: 10.1101/2023.11.28.569126

Figure Lengend Snippet: AOB responses convey stimulus novelty on multiple time scales. (A) GCaMP signal (black line; z-score normalized) during an initial baseline period followed by 18 presentations of stimulus A and 3 presentations of stimulus B presented in a standardized order shown by the boxes below. Vertical dashed red lines indicate investigation events to the less frequently presented stimulus. (B) Pseudocolor plot representing the average GCaMP signal aligned to the start of a sensory investigation. The stimulus order is as shown in panel A and changes between the presented stimulus are represented by white space. Purple asterisks represent novel trials. (C) Left, average GCaMP signal across presentations to rare (stimulus B: red) or frequent (stimulus A: gray) age and sex matched social stimuli. Right, mean GCaMP response to rare stimuli and frequent stimuli. (D) Average AOB GCaMP response to frequent (grey) and rare (red) stimuli during the 20 seconds following stimulus investigation (error bars: SEM). (E) Left, average GCaMP signal across presentations to novel (first presentation of either stimulus A or B: purple; asterisks in A,B, D) or familiar (subsequent presentations of either stimulus A or B: gray) age and sex matched social stimuli. Right, mean GCaMP response to novel (N) stimuli and familiar (F) stimuli.

Article Snippet: We injected a conditional GCaMP expressing AAV virus (AAV9:FLEX:GCaMP6s; Addgene Plasmid #:100845; ) in the AOB (Bregma 3.5, Lateral 1.0, Depth 1.0) of Tbet-cre mice to target M/T cells in the AOB.

Techniques:

Journal: bioRxiv

Article Title: Neural responses to social cues in the accessory olfactory bulb are altered by context and experience

doi: 10.1101/2023.11.28.569126

Figure Lengend Snippet: Analysis pipeline for fiber photometry signals and behavior. (A) GCaMP and isosbestic signal are collected and synced with behavior (left). GCaMP and isosbestic signals are separated utilizing lock-in sampling and amplification (middle). GCaMP and isosbestic signals are compared via regression normalization allowing the isolation of calcium dependent AOB signals from movement. (B) Scored behavioral states and events are synced with calcium-dependent AOB signal z-scores.

Article Snippet: We injected a conditional GCaMP expressing AAV virus (AAV9:FLEX:GCaMP6s; Addgene Plasmid #:100845; ) in the AOB (Bregma 3.5, Lateral 1.0, Depth 1.0) of Tbet-cre mice to target M/T cells in the AOB.

Techniques: Sampling, Amplification, Isolation

Journal: bioRxiv

Article Title: Visual experience instructs the organization of cortical feedback inputs to primary visual cortex

doi: 10.1101/2022.10.12.511901

Figure Lengend Snippet: A ) Schematic illustration of the rearing protocols in the three groups of mice. B ) Schematic of the dual-color imaging configuration. C ) Schematic illustration of the experimental setup. The beams from two lasers were aligned and scanned together while independently controlling their power. D ) Left, fiuorescent image of the cranial window showing the injection site in LM. Right, visual field sign map obtained from intrinsic signal imaging. Light orange circle indicates injection site in LM. E ) Coronal histological section of the injection site in visual cortex. The section was registered to the mouse brain atlas to estimate area boundaries. F ) Image of a region of V1 showing GCaMP6s-expressing axons from LM and jRGECO1a-expressing cells in V1. G ) Two photon field of view of an example plane of GCaMP6s-expressing boutons (top) and jRGECO1a-expressing neurons (bottom). Data in panels D-G is from the same mouse. H ) RF of an example LM bouton and a V1 neuron from the same session. Traces, mean fiuorescence signals for stimulus at each of the locations stimulated. Stimulus onset was at the beginning of each trace and lasted for 1.2 s. Grayscale color map, mean response 0-2 s after stimulus onset. Ellipses, fitted RFs. I ) Visual coordinates of individual LM boutons’ RF center as a function of the mean RF centers of V1 neurons for each imaging session. Blue squares, mean values of the RF centers of LM boutons for each imaging location. Blue lines, linear fits of the mean values (statistics of linear fits in azimuth and elevation; DRP0: t 43 =14.1, p=9e -18 , t 43 =13.4, p=6e -17 , n=45 imaging locations, N=6 mice; DRP21: t 60 =36.7, p=8e -43 , t 60 =24.1, p=1e -32 , n=62, N=9; NR: t 60 =14.9, p=7e -18 , t 40 =10.5, p=5e -13 , n=42, N=6). Identity line is dashed. J ) The relative retinotopic position (ΔRF) was defined as the difierence between the bouton’s RF center (green dot) and the mean RF center of all the V1 neurons for that imaging session (red dot). is the length of the ΔRF vector and θ is the deviation angle. K ) 2D histogram of the distribution of ΔRF for boutons across the three visual experience groups. DRP0; n=12597 boutons, N= 6 mice; DRP21, n=22517 boutons, N=9 mice; NR, n=13250 boutons, N=6 mice. The x indicates the origin. White circles correspond to difierent distances from the origin.

Article Snippet: Virus expressing GCaMP6s ( )(AAV1-Syn-flex-GCaMP6s-WPRE-SV40, Addgene #100843) was injected (50 nL total, 10 nL/min, 250 and 550 μm deep) into the center of LM, using beveled glass pipettes (Drummond) with diameters of 8-12 μm.

Techniques: Imaging, Injection, Expressing, Plasmid Preparation

Journal: Cell reports

Article Title: Cerebellar nuclei neurons projecting to the lateral parabrachial nucleus modulate classical fear conditioning.

doi: 10.1016/j.celrep.2023.112291

Figure Lengend Snippet: Figure 1. DCN neurons project to the lPBN (A) Viral injection of AAV-hSyn-EYFP into the DCN. Left: EYFP expression in the DCN. Right: EYFP signals in the lPBN. Scale bars: 500 mm (left) and 200 mm (right). (B) Retrograde tracing from the lPBN to DCN. Left: strategy for labeling lPBN-projecting DCN neurons. Right: representative images of retro bead injection into the lPBN and retrogradely labeled DCN neurons. FN, fastigial nucleus; IpN, interpositus nucleus; DN, dentate nucleus. Scale bars: 1 mm (top) and 500 mm (bottom). (C) Number of labeled cells along the rostro-caudal axis (n = 3 mice). (D) Whole-cell patch-clamp recordings of optogenetically evoked EPSCs (oEPSCs) in the lPBN via stimulation of ChR2-expressing cerebellar axons. Of 49 cells recorded, 37 cells exhibited time-locked synaptic responses to blue laser stimulation. (E) Example recording traces of oEPSCs with pharmacological treatment. (F) oEPSCs recorded in the lPBN treated with TTX, 4-AP, and NBQX (n = 5 slices from 5 mice; ACSF vs. +TTX, two-tailed paired t test, ***p = 0.0002; +TTX vs. +4- AP, two-tailed paired t test, **p = 0.0036; +4-AP vs. +NBQX, two-tailed paired t test, **p = 0.0088). (G) Anterograde labeling from the DCN to lPBN. AAV8-EF1a-mCherry-IRES-WGA-Cre and AAV1-EF1a-DIO-EYFP were injected into the DCN and the lPBN, respectively. The anterograde transneuronal tracer wheat germ agglutinin (WGA) fused to Cre recombinase in the DCN permits EYFP expression in lPBN neurons receiving input from the DCN. (H) Representative images of DCN-connected lPBN projections. BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CeA, central amygdala; LH, lateral hypothalamic area; PAG, periaqueductal gray; PVH, paraventricular nucleus of the hypothalamus; GPi, globus pallidus internus; ZI, zona incerta; 3V, third ventricle. Scale bar: 500 mm. (I) Anterograde labeling from the DCN to lPBN using synaptophysin-mRuby. AAV1-hSyn-Cre and AAV1-hSyn-DIO-synaptophysin-mRuby were injected into the DCN and the lPBN, respectively. (J) Representative images of synaptophysin-mRuby signals of the DCN-connected lPBN projections. Scale bar: 500 mm. Data are presented as mean ± SEM. See also Figures S1 and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit monoclonal anti-c-Fos (9F6) Cell Signaling Technology Cat# 2250; RRID: AB_2247211 Rabbit polyclonal anti-PACAP-38 Peninsula Laboratories Cat# T-4473; RRID: AB_519166 Sheep polyclonal anti-FoxP2 R&D Systems Cat# AF5647; RRID: AB_2107133 Mouse monoclonal anti-CGRP Abcam Cat# ab81887; RRID: AB_1658411 Alexa Fluor 568-conjugated donkey anti-mouse Invitrogen Cat# A10037; RRID: AB_2534013 Alexa Fluor 594-conjugated donkey anti-sheep Abcam Cat# ab150180; RRID: AB_2716768 Alexa Fluor 568-conjugated goat anti-rabbit Invitrogen Cat# A11011; RRID: AB_143157 Alexa Fluor 647-conjugated donkey anti-rabbit Invitrogen Cat# A31573; RRID: AB_2536183 Bacterial and virus strains AAV-EF1ɑ-DIO-EYFP Addgene Cat# 27056-AAV1; RRID: Addgene_27056 AAV-EF1ɑ-DIO-eNpHR3.0-EYFP Addgene Cat# 26966-AAV1; RRID: Addgene_26966 AAV-EF1ɑ-double floxedhChR2(H134R)-EYFPWPRE-HGHpA Addgene Cat# 20298-AAV1; RRID: Addgene_20298 AAVrg-EF1ɑ-mCherry-IRES-Cre Addgene Cat# 55632-AAVrg; RRID: Addgene_55632 AAV-hSyn-double floxedhChR2(H134R)-EYFP Addgene Cat# 26973-AAV1; RRID: Addgene_26973 AAV-hSyn-eNpHR3.0-EYFP Addgene Cat# 26972-AAV5; RRID: Addgene_26972 AAV-CAG-FLEX-GCaMP6s-WPRE-SV40 Addgene Cat# 100842-AAV1; RRID: Addgene_100842 AAVrg-hSyn-EGFP Addgene Cat# 50465-AAVrg; RRID: Addgene_50465 AAV-hSyn-EGFP Addgene Cat# 50465-AAV5; RRID: Addgene_50465 pENN.AAV-hSyn-Cre-WPRE-hGH Addgene Cat# 105553-AAV1; RRID: Addgene_105553 AAV-hSyn-FLEx-mGFP-2ASynaptophysin-mRuby Addgene Cat# 71760-AAV1; RRID: Addgene_71760 rAAV8-CAG-mCherry-IRES-WGA-Cre UNC vector Core Cat# AV5901FG Chemicals, peptides, and recombinant proteins Normal Donkey Serum Sigma Cat# D9663 Triton X-100 Sigma Cat# X100 DAPI mounting solution Vectorlab Cat# H-1200 Retrobead (red) LumaFluor N/A (Continued on next page) 14 Cell Reports 42, 112291, April 25, 2023

Techniques: Injection, Expressing, Retrograde Tracing, Labeling, Patch Clamp, Two Tailed Test