projection neuron  (Nusbaum Inc)

Journal: eLife

Article Title: Synaptic connectivity of sensorimotor circuits for vocal imitation in the songbird

doi: 10.7554/eLife.104609

Figure Lengend Snippet: ( A ) (left) Parasagittal schematic of the zebra finch brain illustrating HVC afferents (grey) from nucleus interface of the nidopallium (NIf), nucleus avalanche (Av), medial magnocellular nucleus of the anterior nidopallium (mMAN) and nucleus Uvaeformis (Uva), and the three classes of HVC-PNs: HVC X (projecting to Area X, cyan), HVC RA (projecting to RA, magenta), and HVC Av (projecting to Av, yellow). (right) Schematic illustrating the known synaptic connectivity of HVC’s input and output pathways (interneurons schematized in black). ( B ) (top) Schematic of the workflow including opsin expression in afferent areas, retrograde tracer injection in afferent areas and ,whole cell patch-clamp recording of light-evoked currents in acute brain slices. (bottom) Sample image of retrogradely labeled HVC-PN classes in a brain slice used for patch-clamp recordings (scalebar 100 µm). ( C ) Schematic of whole-cell recording of light-evoked synaptic currents in HVC-PNs receiving monosynaptic inputs from one of the afferent areas expressing eGTACR1 (red), as well as polysynaptic inputs from local HVC-PNs (white circle) and interneurons (black circle). Recordings are performed at holding potential (Vh)=+10 mV and –70 mV. Sample traces report the effect of bath application of DNQX (green) and gabazine (GBZ, purple), which suppress oEPSC and oIPSC, respectively. Glutamatergic monosynaptic currents are pharmacologically isolated by bath application of TTX +4AP: sample traces (average of 20 sweeps) portray a typical case of a cell displaying oEPSC with a monosynaptic component (inset shows 20 oEPSCs sweeps in grey, averaged in the thick traces in black and blue), and polysynaptic oIPSC, as 4AP (blue) application results in a partial restoration of oEPSC, but not of oIPSC (red lines represent light stimuli, 1ms; scalebar: 100ms, 100 pA).

Article Snippet: NIf neurons projecting to HVC (NIf HVC ) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 ( ; ).

Techniques: Expressing, Injection, Patch Clamp, Labeling, Slice Preparation, Isolation

Journal: eLife

Article Title: Synaptic connectivity of sensorimotor circuits for vocal imitation in the songbird

doi: 10.7554/eLife.104609

Figure Lengend Snippet: ( A ) (left) Schematic of the experimental timeline, illustrating injection of AAV-eGtACR1 in Uva, followed by retrograde tracer injections in HVC efferent areas and whole-cell patch-clamp recording in acute brain slices; (right) sample image of eGtACR1-mScarlet expression in Uva (scalebar 200 µm). ( B ) (top) pie charts representing the likelihood of observing oEPSCs in HVC X (cyan), HVC RA (magenta), or HVC Av (yellow) (Fisher’s exact test, p<0.001). Numbers in the pie fragments represent the number of cells in which current (colored) or no current (white) was found. Numbers next to the pie charts represent the number of animals from which the data is obtained. (bottom) bar chart representing the number of cells where both oEPSCs and oIPSCs could be elicited (black), only oEPSCs but no oIPSCs (grey lines), only oIPSCs but no oEPSCs (grey checkers), or neither (white), for subsets of cells from the HVC-PN classes’ pie charts aligned above (Fisher’s exact test, p=0.0244). ( C ) Violin and scatter plot and sample traces reporting average measured oEPSC amplitude of each cell, by cell class (Kruskal-Wallis test H(2)=2.241, p=0.3262; n=cells (animals); red lines represent light stimuli, 1ms; scalebars, 100ms, 100 pA). ( D ) Violin and scatter plot and sample traces of average measured oIPSC amplitude of each cell, by cell class (H(2)=0.5946, p=0.7428; scalebars, 100ms, 100 pA). ( E ) Violin and scatter plot of the ratio of oEPSC and oIPSC peak amplitude of each cell where both are measured and ≠0, per cell class (H(2)=0.2716, p=0.2572). ( F ) (top) sample traces and plot representing the amplitude of post-synaptic currents evoked by lightly-driven release of neurotransmitter from Uva axonal terminals in HVC; oEPSCs amplitudes are reported before (HVC X cyan, HVC RA magenta, HVC Av yellow) and after bath application of TTX (black) and 4AP (grey, green outline indicates polysynaptic oEPSC, see methods), (n=cells (animals); red lines represent light stimuli, 1ms; scalebars, 100ms, 100 pA) (bottom) bar charts representing the likelihood of observing polysynaptic oEPSCs in HVC X (cyan), HVC RA (magenta), or HVC Av (yellow) (data from panel B), and (grey) likelihood of a subset of the corresponding oEPSCs to be monosynaptic. ( G ) Sample images reporting retrogradely labeled HVC-projecting neurons in UVA (cyan, white circles) together with in situ labeling of glutamatergic (SLC17A6, yellow, white arrowheads) and GABAergic (GAD1, magenta, gray arrowheads) markers transcripts (scalebar 200 µm, inset 20 µm).

Article Snippet: NIf neurons projecting to HVC (NIf HVC ) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 ( ; ).

Techniques: Injection, Patch Clamp, Expressing, Labeling, In Situ

Journal: eLife

Article Title: Synaptic connectivity of sensorimotor circuits for vocal imitation in the songbird

doi: 10.7554/eLife.104609

Figure Lengend Snippet: ( A ) (left) Schematic of the experimental timeline, illustrating injection of AAV-eGtACR1 in NIf, followed by retrograde tracer injections in HVC efferent areas and whole-cell patch-clamp recording in acute brain slices; (right) sample image of eGtACR1-mScarlet expression in NIf (scalebar 200 µm). ( B ) (top) pie charts representing the likelihood of observing oEPSCs in HVC X (cyan), HVC RA (magenta) or HVC Av (yellow) (Fisher’s exact test, p=0.2783). Numbers in the pie fragments represent the number of cells in which current (colored) or no current (white) was found. Numbers next to the pie charts represent the number of animals from which the data is obtained. (bottom) bar chart representing the number of cells where both oEPSCs and oIPSCs could be elicited (black), only oEPSCs but no oIPSCs (grey lines), only oIPSCs but no oEPSCs (grey checkers), or neither (white), for subsets of cells from the HVC-PN classes’ pie charts aligned above (Fisher’s exact test, p=0.5841). ( C ) Violin and scatter plot and sample traces reporting average measured oEPSC amplitude of each cell, by cell class (Kruskal-Wallis test, H(2)=6.135, p=0.0465; n=cells (animals); red lines represent light stimuli, 1ms; scalebars, 100ms, 100 pA). ( D ) Violin and scatter plot and sample traces of average measured oIPSC amplitude of each cell, by cell class (H(2)=6.182, p=0.0455; scalebars, 100ms, 100 pA). ( E ) Violin and scatter plot of the ratio of oEPSC and oIPSC peak amplitude of each cell where both are measured and ≠0, per cell class (H(2)=3.305, p=0.1916). ( F ) (top) sample traces and plot representing the amplitude of post-synaptic currents evoked by lightly-driven release of neurotransmitter from NIf axonal terminals in HVC; oEPSCs amplitudes are reported before (HVC X cyan, HVC RA magenta, HVC Av yellow) and after bath application of TTX (black) and 4AP (grey, green outline indicates polysynaptic oEPSC, see Materials and methods), (n=cells (animals); blue lines represent light stimuli, 1ms; scalebbars, 100ms, 100 pA) (bottom) bar charts representing the likelihood of observing polysynaptic oEPSCs in HVC X (cyan), HVC RA (magenta) or HVC Av (yellow) (data from panel B), and (grey) likelihood of a subset of the corresponding oEPSCs to be monosynaptic. ( G ) Sample images reporting retrogradely labeled HVC-projecting neurons in NIf (cyan, white circles) together with in situ labeling of glutamatergic (SLC17A6, yellow, white arrowheads) and GABAergic (GAD1, magenta, gray arrowheads) markers transcripts (scale bar 200 µm, inset 20 µm).

Article Snippet: NIf neurons projecting to HVC (NIf HVC ) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 ( ; ).

Techniques: Injection, Patch Clamp, Expressing, Labeling, In Situ

Journal: eLife

Article Title: Synaptic connectivity of sensorimotor circuits for vocal imitation in the songbird

doi: 10.7554/eLife.104609

Figure Lengend Snippet: ( A ) Schematic of the known mMAN afferent and efferent circuitry, together with the proposed new connection towards Av (red dashed arrow), and schematized injection of retrograde tracers in HVC and Av. ( B ) Sample images of retrogradely labeled mMAN cells projecting to HVC (magenta) or to Av (cyan), and merged image. Insets report magnified selection (dashed square box in the images) illustrating the potential three subpopulations identified: mMAN HVC (white arrowheads), mMAN Av (grey arrowheads) and mMAN HVC+Av (overimposed white and grey arrowheads) (scalebars: 100 µm, inset: 10 µm). ( C ) Bar chart displaying the number of retrogradely mMAN HVC , mMAN Av , and mMAN HVC+Av labeled cells, in three birds (averaged across hemispheres, 2–6 slices/bird). ( D ) BDA labeling of anterograde projection to Av from mMAN, inset magnifies the terminal field in Av (scale bars: 200 µm, inset: 50 µm).

Article Snippet: NIf neurons projecting to HVC (NIf HVC ) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 ( ; ).

Techniques: Injection, Labeling, Selection

Journal: eLife

Article Title: Synaptic connectivity of sensorimotor circuits for vocal imitation in the songbird

doi: 10.7554/eLife.104609

Figure Lengend Snippet: ( A ) Table reporting descriptive statistics relative to oEPSCs and oIPSCs as reported in . ( B ) Violin and scatter plots representing oPSC rise latencies reported in gray line marks the minimal latency (1st quartile) to oPSC rise across the cell types, for ease of comparison (Mixed-effects analysis, oEPSC vs. oIPSC F(1,35)=8.439, p=0.0063, HVC RA p=0.0198; HVC PN F(2,64)=0.7545, p=0.4744); ( C ) oPSCs rise latency for cells where both oEPSC and oIPSC were measured (2 W ANOVA, oEPSC vs. oIPSC F(1,35)=8.294, p=0.0067, HVC RA p=0.0470); ( D ) relative delay of oIPSC compared to oEPSC across the cells reported in ( C ) (Kruskal-Wallis test, H=0.2028, p=0.9036). ( E ) Sample images reporting retrogradely labeled HVC-projecting neurons in mMAN (cyan, white circles) together with in situ labeling of glutamatergic (SLC17A6, yellow, white arrowheads) and GABAergic (GAD1, magenta, gray arrowheads) markers transcripts (scale bar 200 µm, inset 20 µm).

Article Snippet: NIf neurons projecting to HVC (NIf HVC ) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 ( ; ).

Techniques: Comparison, Labeling, In Situ

Journal: eLife

Article Title: Synaptic connectivity of sensorimotor circuits for vocal imitation in the songbird

doi: 10.7554/eLife.104609

Figure Lengend Snippet: ( A ) Table reporting descriptive statistics relative to oEPSCs and oIPSCs as reported in . ( B ) Violin and scatter plots representing oPSC rise latencies reported in (gray line marks the minimal latency (1st quartile) to oPSC rise across the cell types, for ease of comparison (Mixed-effects analysis, oEPSC vs. oIPSC F(1,39)=21.78, p<0.001, HVC RA p<0.001, HVC Av p=0.0377; HVC PN F(2,70)=2.764, p=0.0699); ( C ) oPSCs rise latency for cells where both oEPSC and oIPSC were measured (2 W ANOVA, oEPSC vs. oIPSC F(1,39)=14.26, p<0.001, HVC RA p<0.001); ( D ) relative delay of oIPSC compared to oEPSC across the cells reported in C) (Kruskal-Wallis test, H=6.403, p=0.0407). ( E ) Sample images reporting retrogradely labeled HVC-projecting neurons in Av (cyan, white circles) together with in-situ labeling of glutamatergic (SLC17A6, yellow, white arrowheads) and GABAergic (GAD1, magenta, gray arrowheads) markers transcripts (scalebar 200 µm, inset 20 µm).

Article Snippet: NIf neurons projecting to HVC (NIf HVC ) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 ( ; ).

Techniques: Comparison, Labeling, In Situ

Journal: eLife

Article Title: Synaptic connectivity of sensorimotor circuits for vocal imitation in the songbird

doi: 10.7554/eLife.104609

Figure Lengend Snippet: ( A ) Schematic representing a simplified version of the long-range connectivity map for song. Colors represent the anatomical location of each projection: gray: DVR, red: basal ganglia, green: thalamus, purple: midbrain. HVC afferents described in this manuscript are highlighted by large arrows. The novel mMAN-Av projection is highlighted by a dashed outline. ( B ) Schematic of the HVC afferent connectivity map resulting from the present work, complemented with projections between HVC afferent areas (on the left) and between HVC projection neurons as per previous reports (on the right). For conceptualization purposes, afferent connectivity to HVC-PNs is shown only when the rate of monosynaptic connectivity reaches 50% of neurons examined: NIf monosynaptically contacts all three HVC-PNs, while Uva is preferentially monosynaptically connected to HVC RA , mMAN to HVC X and HVC Av , and Av to HVC X . ( C ) Sankey diagram displaying the prevalence of connectivity for each input and cell subtype combination, based on polysynaptic and monosynaptic connectivity rates described in – and – .

Article Snippet: NIf neurons projecting to HVC (NIf HVC ) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 ( ; ).

Techniques:

Journal: eLife

Article Title: Synaptic connectivity of sensorimotor circuits for vocal imitation in the songbird

doi: 10.7554/eLife.104609

Figure Lengend Snippet: ( A ) Pie charts representing the likelihood of evoking oEPSCs in HVC X , and violin plots representing the oEPSCs amplitude, when stimulating afferents from NIf, UVA, mMAN or Av (probability: Fisher’s exact test, p<0.001; amplitude: Kruskal-Wallis test, H(3)=17.18, p<0.001, NIf vs. UVA p=0.1695, NIf vs. mMAN, p<0.001, NIf vs. Av p=0.0993). ( B ) Same as ( A ) for oIPSCs (probability: Fisher’s exact test, p=0.2828; amplitude: Kruskal-Wallis test, H(3)=5.008, p=0.1712). ( C ) Violin plots reporting the oEPSC rise latency in HVCX upon stimulation of the four afferents (Kruskal-Wallis test, H(3)=11.91, p=0.0077, NIf vs. UVA p=0.0534, NIf vs. mMAN, p=0.0119, NIf vs. Av p=0.0345). ( D ) Same as ( C ) for oIPSCs (Kruskal-Wallis test, H(3)=19.23, p<0.001, NIf vs. UVA p=0.5428, NIf vs. mMAN, p=0.0016, NIf vs. Av p>0.9999, mMAN vs. Av p=0.0028). ( E–H ) Same as ( A-D ) but for HVC RA . ( E ) (probability: Fisher’s exact test, p<0.001; amplitude: Kruskal-Wallis test, H(3)=3.594, p=0.3088). ( F ) (probability: Fisher’s exact test, p=0.0075; amplitude: Kruskal-Wallis test, H(3)=2.132, p=0.5454). ( G ) (Kruskal-Wallis test, H(3)=12.44, p=0.0060, NIf vs. UVA p>0.999, NIf vs. mMAN, p=0.6258, NIf vs. Av p=0.3175, UVA vs. Av p=0.0128). ( H ) (Kruskal-Wallis test, H(3)=6.622, p=0.0850). ( I–L ) Same as (A-D) but for HVC Av . ( I ) (probability: Fisher’s exact test, p=0.3150; amplitude: Kruskal-Wallis test, H(3)=18.46, p<0.001, NIf vs. UVA p=0.0016, NIf vs. mMAN, p=0.0929, NIf vs. Av p=0.0010). ( J ) (probability: Fisher’s exact test, p=0.1869; amplitude: Kruskal-Wallis test, H(3)=12.52, p=0.0058, NIf vs. UVA p=0.1043, NIf vs. mMAN, p>0.999, NIf vs. Av p=0.0405). ( K ) (Kruskal-Wallis test, H(3)=6.441, p=0.0920). ( L ) (Kruskal-Wallis test, H(3)=7.653, p=0.0538).

Article Snippet: NIf neurons projecting to HVC (NIf HVC ) selectively expressed the glutamatergic marker SLC17A6, but not the GABAergic marker GAD1 ( ; ).

Techniques:

Journal: Neurobiology of disease

Article Title: α-Synuclein aggregation decreases cortico-amygdala connectivity and impairs social behavior in mice.

doi: 10.1016/j.nbd.2024.106702

Figure Lengend Snippet: Fig. 4. α-Syn pathology disrupts functional connectivity of the mPFC-BLA synapses. A) Top, diagram showing overall experimental design. Bottom, Representative AAV-ChR2-eYFP injection site in the mPFC (left) and the eYFP-labeled axonal terminal field in the BLA (right). B–C) Decreased amplitude of optogenetically evoked mPFC-BLA EPSCs in PFFs-injected mice relative to controls at both 3 (B) and 6 (C) mpi. Top) Representative traces of EPSCs evoked by optical stimulation intensities of 1.8, 3.6, and 12 mW/mm2. Blue arrows indicate when the optogenetic stimulation was delivered. Bottom) Summarized graphs showing reduced amplitude of mPFC-BLA ESPCs across a range of stimulation intensities in PFFs-injected mice relative to controls at both 3 and 6 mpi. N = 44 cells/7 mice at 3 mpi and 57 cells/7 mice at 6 mpi. Two-way ANOVA followed by Sidak multiple comparisons test. D) Representative traces of mPFC-BLA EPSCs recorded at −80 mV and + 40 mV from both controls and PFFs-injected mice. Red dots indicate where the AMPA receptor- and NMDA receptor-mediated EPSCs were measured at −80 mV and + 40 mV, respectively. Blue dashed lines indicate when optogenetic stimulation was delivered. E-F) Summarized result showing reduced AMPA/NMDA ratios at mPFC-BLA synapses in PFFs-injected mice relative to controls at 3 mpi (E, control = 3.3 [2.7, 5.4], n = 29 cells/4 mice, PFFs = 2.8 [1.8, 4.0], n = 41 cells/7 mice, p = 0.05) and 6 mpi (F, control = 2.7 [1.9, 3.8], n = 28 cells/4 mice, PFFs = 1.9 [1.5, 2.5], n = 46 cells/6 mice, p = 0.0006). G) Representative traces of mPFC-BLA EPSCs evoked by paired optogenetic stimulation pulses at −80 mV in both controls and PFFs-injected mice. Blue arrows indicate when the optogenetic stimulation was delivered. H–I) Summarized results showing reduced ratios of EPSC2/EPSC1 at the mPFC-BLA synapses in PFFs-injected mice relative to controls at both 3 mpi (H, control = 1 [0.75, 1.32], n = 36 cells/5 mice; PFFs = 0.85 [0.67, 1.1], n = 48 cells/7 mice, p = 0.016, MWU) and 6 mpi (I, control = 1.04 [0.85, 1.32], n = 31 cells/4 mice, PFFs = 0.94 [0.72, 1.09], n = 57 cells/7 mice, p = 0.008, MWU test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To optogenetically evoke excitatory postsynaptic currents (EPSCs) of BLA projection neurons, we injected 0.3 μL AAVs encoding ChR2(H134R)-eYFP (titer= 1× 1012 GC/mL, Addgene# 26973, RRID: Addgene_127090) into the layer 5 of the medial prefrontal cortex (mPFC, A-P,+2.1 mm from bregma; M-L, ± 0.3 mm from the midline; D–V, − 2.3 mm from the brain surface) and the centromedial thalamus (A-P, − 1.5 mm from bregma; M-L, 0 mm from the middle; D–V, − 3.3 mm from the brain surface) at a rate of 0.1 μL/min.

Techniques: Functional Assay, Injection, Labeling, Control