Display Settings:

Items per page

Results: 9

1.
Figure 5

Figure 5. RVLM-CA neurons make synapses with DMV cholinergic neurons. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A–D, Electron micrographs of the DMV from DβH-Cre mice injected with DIO-ChR2-mCherry AAV2 in RVLM. mCherry-ir axonal varicosities labeled with immunoperoxidase reaction product make synapses onto dendrites containing gold-silver labeling for ChAT. The postsynaptic density (black arrows), indicate that these synapses are asymmetric and presumably glutamatergic. E, Example of symmetric synapse (white arrows) between mCherry-ir (peroxidase) terminal and ChAT-ir (gold) dendrite. F, Example of mCherry-positive axon containing dense core vesicles (white arrows) reminiscent of catecholaminergic terminals. Scale in E, 0.5 µm applies to A–E. Scale in F, 0.5 µm.

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.
2.
Figure 2

Figure 2. Verification that Fluoro-Gold (FG) labels ChAT neurons in the DMV and that the recorded neurons are vagal premotor neurons. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A1–A3, Photomicrograph of DMV illustrating that ChAT-ir neurons (red) in DMV(A1) are also FG positive (blue) (A2). A3, Merged image of A1 and A2, double labeled neurons appear pink. Scale in A1, 100 µm, applies to A1–A3. B, Example of a slice reacted for immunohistochemistry to reveal the biocytin label (B1) showing the cell is also FG-ir (green, B2). B3, Merge of B1 and B2 showing the cell is within the DMV and also FG, thus projecting outside the brain. Scale in B1, 50 µm, applies to B1–B3. C1, C2, Illustrations of biocytin filled neurons recorded in slices and reconstructed showing the dendrites and axon projecting towards the periphery of the section. Scale in C2, 0.5 mm applies to C1–C2.

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.
3.
Figure 4

Figure 4. Axonal varicosities from C1 neurons in rat DMV contain PNMT and VGLUT2. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A, Photomicrograph showing that injection of AAV2-DIO-EYFP into the RVLM drives expression of EYFP in C1 (PNMT-ir) neurons in TH-Cre rats. EYFP immunoreactivity is in green and phenylethanolamine-N-methyl transferase (PNMT)-ir is in blue. Double labeled neurons appear aqua (arrows). Scale, 50 µm. B,Virtually all adrenergic varicosities (PNMT-ir, blue) within the DMV (B1, denoted by arrows or arrowheads) are also glutamatergic (VGLUT2-ir, red) (B2). The EYFP terminals (green) (B3, arrowheads) originating from C1 RVLM neurons are both adrenergic (red) and glutamatergic (green). B4, Merge of B1–B3. Triple labeled terminals (arrowheads) appear white. Double-label terminals (PNMT + VGLUT2) appear purple (arrows) when the colors are merged. C, Adrenergic terminals in DMV are also glutamatergic in naïve TH-Cre rats (no AAV2 injection). Adrenergic nerve terminals (PNMT-ir, red) located in DMV (C1) are also glutamatergic (VGLUT2-ir, green) (C2), arrows. These terminals appear yellow when the colors are merged in C3 (arrows). Note that practically all the PNMT-ir terminals are also VGLUT2-ir similar to the virus-injected rat shown in B. Scale in B1, 10 µm applies to B–C.

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.
4.
Figure 7

Figure 7. Photostimulation of ChR2-expressing axons produces exclusively EPSCs in DMV neurons. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A1, DMV neuron voltage clamped at VM −79 mV (cesium chloride filled electrode). Top trace EPSC frequency (integrated rate histogram with 0.5 s bin), bottom trace original recording. Solid blue block represents the period of photostimulation. A2, Excerpt of EPSCs recorded during the early part of the after discharge. B1, Same neuron voltage clamped at VM −9 mV. Top trace illustrates the lack of change of IPSC frequency during photostimulation, bottom trace is the original recording. B2, Excerpt of the recording trace obtained during the stimulation period. C, Group data (N= 9 neurons) showing inhibitory an excitatory PSC frequency at baseline (B), during the 20Hz stimulus (S), and after full recovery (R); asterisks (P<0.0001).

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.
5.
Figure 8

Figure 8. EPSCs evoked in DMV neurons are glutamatergic. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A1, Response of a DMV neuron clamped at VM −79 mV to photostimulation (1ms, 20 Hz, 5 s; blue block represents period of photostimulation). From left to right, control response, response following application of bucuculline (bic, 10µM) and strychnine (strych, 20µM) and response after further addition of ionotropic glutamate receptor blockers AP5 (50 µM) and CNQX (10 µM. Bottom traces, original recordings; top traces low-pass filtered traces (5 Hz). A2, Group data (N = 4). Area under the curve (AUC) obtained from the original recording trace. Asterisk indicates statistical significance from both control and bicuculline/strychnine B1, Response of a DMV neuron clamped at VM −79 mV to photostimulation (1ms, 20 Hz, 5 s). From left to right, control response, response following application of kynurenate (kyn, broad-spectrum ionotropic glutamate receptor antagonist, 1mM). Bottom traces, original recordings; top traces low-pass filtered traces (5 Hz). B2, Group data (N = 7). AUC obtained from the original recording traces. Asterisk (P<0.05) indicates statistical significance from both control and recovery period.

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.
6.
Figure 9

Figure 9. Photostimulation of ChR2-expressing fibers activate DMV neurons after action potential blockade. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A, EPSC evoked in a DMV neuron (VM: −79 mV) by 1 ms light pulses at 0.5 Hz (average of 75 consecutive stimuli) before any drug (left trace), following tetrodotoxin application (TTX, 1µM), after addition of 4-aminopyridine (TTX + 4-AP, 100µM) and in the presence of TTX, 4-AP and glutamatergic antagonists CNQX and AP5. Blue arrowheads indicate time of laser onset. B, Group data (N= 6 neurons). Asterisks indicate significant differences between groups joined by horizontal bars. C, Experiment illustrating the effect of the same sequence of drug application on the response of a DMV neuron to a train of high frequency photostimulation (1 ms, 20 Hz, 5 s). Solid blue block indicates period of photostimulation. D, Group data (N= 8). Asterisks indicate significant differences between groups joined by horizontal bars.

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.
7.
Figure 1

Figure 1. RVLM-Catecholaminergic (RVLM-CA) neurons innervate the dorsal motor nucleus of the vagus (DMV) in DβH-Cre mice. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A, Rostrocaudal distribution of mCherry+TH-ir neurons (average of 5 cases). The ChR2-expressing neurons were confined to the RVLM. FN, caudal extent of the facial motor nucleus provided as reference. Dual-labeled neurons appear orange-yellow. Untransfected TH-ir neurons appear green. B, Photomicrograph of the RVLM one month after injection of AAV2-DIO-ChR2-mCherry into the same region in a DβH-Cre mice. mCherry immunoreactivity (red) is detectable exclusively in tyrosine hydroxylase (TH)-immunoreactive(ir) neurons (green). Scale, 100 µm. C, Photomicrograph showing the dense plexus of nerve terminals arising from mCherry-ir RVLM-CA neurons. ChR2-mCherry is visualized with the nickel-DAB method resulting in a black color. Choline acetyl-transferase (ChAT)-immunoreactivity is visualized with DAB resulting in a brown color. Note the dense terminal field in the DMV (10) that avoids the hypoglossal nucleus (12). cc, central canal. Scale, 200 µm. D, Higher power photomicrograph of the DMV innervation seen in C showing fibers and synaptic boutons arising from ChR2-expressing RVLM-CA neurons in close proximity of the DMV cholinergic neurons. Scale, 30 µm.

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.
8.
Figure 3

Figure 3. VGLUT2 is present in DMV varicosities that originate from RVLM-CA neurons but is absent from those that emanate from A1 noradrenergic and raphe serotonergic neurons. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A, Drawing of mouse brain coronal hemisection at ~7.64 mm caudal to bregma. The red box represents the region of the DMV where axonal varicosities were counted and the photomicrographs were taken. Scale in A, 500 µm. B1, Photomicrograph of mCherry-ir terminals (red) in the DMV from a DβH-Cre mouse injected in RVLM with DIO-ChR2-mCherry AAV2. B2, TH-ir varicosities within the same field. B3, Merged image (B1 + B2) showing that all the mCherry-ir varicosities are yellow, i.e. contain TH. C, Same experiment as in B but revealing mCherry with a rabbit secondary tagged with Dylight 649 and pseudocolored blue (C1) and both TH-ir (green, C2) and VGLUT2-ir varicosities (red, C3). C4, Merged image (C1 + C2 + C3) showing triple labeled terminals (appearing white, arrowheads) as well as terminals double labeled only for TH and VGLUT2 (appearing yellow, arrows). D1, Photomicrograph of mCherry-ir terminals (red) in the DMV from a DβH-Cre mouse injected in RVLM with DIO-ChR2-mCherry AAV2. D2, VGLUT2-ir varicosities within the same field. D3, Merged image (B1 + B2) showing that all the mCherry-ir varicosities are yellow, i.e. contain VGLUT2. E, Similar experiment to B except the DβH-Cre mouse was injected in the A1 region (caudal VLM). E1, mCherry E2, VGLUT2 E3, Merge of C1 + C2. Note that the mCherry-ir varicosities are red, i.e. are not VGLUT2-ir. F, Similar experiment to B except the AAV2 was injected into the raphe obscurus of an ePet-Cre mouse. F1, mCherry F2, VGLUT2 F3, Merge of F1 + F2. Note that the mCherry-ir varicosities are red, i.e. are not VGLUT2-ir. G, Similar experiment to B except the AAV2 was injected into the RVLM of a DβH-Cre(Cre/0); Vglut2(flox/flox) mouse (DβH -VGKO). G1, mCherry G2, VGLUT2 G3, Merge of G1 + G2. Note that the mCherry-ir varicosities are red, i.e. are not VGLUT2-ir. Abbreviations: 10, dorsal motor nucleus of the vagus; 12, hypoglossal motor nucleus; AP, area postrema; cc, central canal; DβH, dopamine beta hydroxylase; ION, inferior olivary nucleus; NTS, nucleus of the solitary tract; st, solitary tract. Scale in G3 applies to B–G, 10 µm.

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.
9.
Figure 6

Figure 6. Photostimulation of ChR2-labeled axons produces PSCs in DMV neurons and increases their discharge rate. From: Glutamatergic neurotransmission between the C1 neurons and the parasympathetic preganglionic neurons of the dorsal motor nucleus of the vagus.

A1, Compound PSC (note variable latency) evoked in a DMV neuron clamped at −79 mV by nine consecutive 1 ms light flashes (473 nm) delivered at 0.5 Hz. The red trace denotes a failure. Blue arrowhead and line indicates time of laser onset throughout figure. A2, Event-triggered average of the PSCs evoked in the cell shown in A1 (75 stimuli). B1, Compound PSC evoked in another DMV neuron (VM −79 mV) by nine consecutive 1 ms light flashes (473 nm) delivered every 2 seconds. This second example illustrates a larger and more multiphasic response. B2, Event-triggered average of the EPSC evoked in the cell shown in B1 (75 stimuli). B3, The inset shows the EPSC after-discharge that follows the large initial EPSC and the main graph is a peri-event histogram depicting the probability of occurrence of EPSCs following the laser flashes. The red line is a single exponential with a time constant of 0.6s that fits the kinetics of the after-discharge with a probability of 0.9. C1, Top trace: integrated rate histogram of the PSC frequency before during and after photostimulation (20 Hz, 1 ms, train duration 5 s, laser on time represented by solid blue block). Second trace from top, original recording obtained from DMV neuron clamped at VM −79 mV. Lower traces, higher resolution excerpts from the original recording. Note that, during high frequency photostimulation, the evoked PSCs are asynchronous with respect to the light pulses. Solid blue block indicates period of photostimulation. C2, Event-triggered average of the PSCs evoked by photostimulation (100 light pulses). Note that the trace is flat indicating asynchrony between PSCs and light pulses. D, Group data showing the average kinetics of the after-discharge (N= 16 neurons). Asterisks indicate p< 0.001 between group and baseline except 30 s, p<0.05. E1, Response of a DMV neuron to photostimulation of RVLM catecholaminergic neuron input (1ms, 20 Hz, 5s). Bottom trace, original current clamp recording (−10 pA bias current injected to silence the cell). Middle trace, first differential of original trace to highlight PSPs. Top trace, PSP frequency (integrated rate histogram, 1 s bin). Period of photostimulation is indicated by solid blue block. E2, High resolution excerpt illustrating (from the bottom up) the original recording, the first differential used to detect PSPs and analyze their frequency (dotted line, detection threshold) and the detected PSPs.

Seth D. DePuy, et al. J Neurosci. ;33(4):1486-1497.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk