ChR2-mCherry labeled terminals originating from RVLM-catecholaminergic (RVLM-CA) neurons innervate the dorsal motor nucleus of the vagus (DMV) and contain VGLUT2
The location of the neurons that expressed ChR2-mCherry following injection of DIO-ChR2-mCherry AAV2 was examined in five mice that were also used for ultrastructural analysis of labeled terminals within the DMV. Consistent with our previous study in which we utilized the same electrophysiologically guided procedure to inject the vector into the rostral portion of the ventrolateral medulla (RVLM) (Stornetta et al., 2012
), the mCherry-immunoreactive (ir) neurons were confined to the RVLM, mostly caudal to the facial motor nucleus (). The mCherry-positive neurons were 97% ± 1% tyrosine hydroxylase (TH)-ir, hence potentially catecholaminergic ().
RVLM-Catecholaminergic (RVLM-CA) neurons innervate the dorsal motor nucleus of the vagus (DMV) in DβH-Cre mice
In all mice, the DMV contained a dense network of mCherry-ir terminals that made close appositions with choline acetyl-transferase (ChAT)-ir, i.e. cholinergic neurons ().
Evidence that neurons recorded in the DMV were parasympathetic preganglionic cells
We confirmed that these DMV cholinergic neurons were parasympathetic preganglionic neurons by showing that all these cholinergic neurons contained FG in mice that had received an intraperitoneal (ip) injection of this retrogradely transported dye. After i.p. injection of FG in 3 animals, neurons labeled with FG within the DMV were counted and the proportion of FG-labeled DMV neurons that were immunoreactuve for choline-acetyl transferase (three sections per mouse) was determined. The coincidence was 95.1 ± 1.5%, (total of 1219 neurons counted, example in ). Brainstem slices were made from an additional 3 mice injected i.p. with FG and 8 FG-positive DMV neurons cells were recorded and filled with biocytin as described (example in ). The extensive filling with biocytin allowed for computer-assisted reconstruction of the dendrites and axonal processes of four cells (2 examples shown in ) showing a long, presumably axonal, process exiting along the expected trajectory for a parasympathetic preganglionic neuron.
Verification that Fluoro-Gold (FG) labels ChAT neurons in the DMV and that the recorded neurons are vagal premotor neurons
Since almost all mCherry-expressing cells in the RVLM of DIO-ChR2-mCherry AAV2 injected animals were TH-ir, we expected that a similarly high proportion of the mCherry-ir terminals located in the DMV would also be TH-ir but the ChR2-containing axons present in the DMV could conceivably have originated primarily from the very few TH-negative hence apparently non-catecholaminergic neurons that expressed the transgene. To exclude this possibility, in three DβHCre/0 mice injected with AAV2, coronal sections were reacted for simultaneous immunofluorescent detection of TH and mCherry and we counted singly (mCherry-ir) and dually-labeled axonal varicosities (both mCherry and TH-ir) in the region of the DMV outlined in , which also corresponds to the region that was explored electrophysiologically. In this region, most (98.6 ± 0.2%) of the mCherry-positive axonal varicosities were TH-ir (; 1370 total terminals counted in 3 sections, one section per mouse). Parallel experiments were done to determine what proportion of the ChR2-expressing axonal varicosities contained VGLUT2 immunoreactivity in the same region of the DMV. Most (97 ± 1 %) mCherry-ir axonal varicosities were also VGLUT2-ir (; 1950 total terminals counted in 3 sections, one section per mouse). On statistical grounds, this evidence demonstrates that most ChR2 axonal varicosities contain both VGLUT2 and TH immunoreactivities. By performing triple-label experiments, we were able to find direct evidence of the presence of all three markers (mCherry, VGLUT2 and TH) within the same terminals in the DMV although the degree of coincidence was not quantified ().
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
Control experiments were done to verify that we correctly identified the presence of VGLUT2 in the terminals of the RVLM-CA neurons and to exclude the possibility that the presence of VGLUT2 might be an artifact caused by AAV2 transduction. In 3 DβHCre/0
mice, DIO-ChR2-mCherry AAV2 was injected more caudally within the ventrolateral medulla where the A1 group of noradrenergic neurons resides. In rats, very few A1 neurons contain VGLUT2 mRNA suggesting that they are not glutamatergic (Stornetta et al., 2002
). The cell bodies of these caudal VLM catecholaminergic neurons expressed high levels of transgene (not shown). These cells also projected to the dorsal vagal complex and adjacent nucleus of the solitary tract but very few of their axonal varicosities (8.7 ± 1.5 %) were also VGLUT2-ir (; 567 total terminals counted in one section each from 3 mice). In the second control group, we injected the same amount of DIO-ChR2-mCherry AAV2 into the raphe obscurus of three ePet Cre/0
mice, a procedure that results in selective expression of the transgene by serotonergic neurons (Scott et al., 2005
; Depuy et al., 2011
). These serotonergic neurons also abundantly innervated the dorsal vagal complex. Most of their varicosities were not VGLUT2-ir (). Only 7.4 ± 1% of the mCherry-ir terminals originating from these raphe neurons appeared VGLUT-ir (1137 terminals counted from one section each of 3 mice). The third control group consisted of 3 DβH-CreCre/0
mice that received the standard injections of DIO-ChR2-mCherry AAV2 in the C1 region. The DMV of these mice contained approximately the same density of mCherry-ir axonal projections as the control (DβHCre/0
) mice but only 8.2 ± 3.2% of the mCherry varicosities were also VGLUT2-ir (). In summary, within the mouse dorsal motor nucleus of the vagus, the axonal varicosities of the RVLM-CA neurons (presumptive C1 cells) typically contained both TH and VGLUT2, whereas very few terminals from A1 noradrenergic and raphe obscurus serotonergic neurons exposed to the same AAV2 contained VGLUT2. The absence of VGLUT2 in serotonergic neurons conforms to expectation since this particular vesicular transporter is not known to be expressed by these neurons in adult tissue (Gras et al., 2002
). The small number of VGLUT2-positive terminals originating from A1 and raphe could conceivably be false positive results caused by the close juxtaposition of two terminals from different neurons and likely represents the “background”. Finally, we demonstrated the selectivity of the immunohistochemical detection of VGLUT2 within the varicosities emanating from RVLM-CA cells by showing that VGLUT2 immunoreactivity was present only at “background” level when the Cre-dependent vector was injected into DβHCre/0
The final light microscopy histological experiments were conducted in TH-Cre rats and had three goals. One was to test whether the projection from RVLM-CA neurons to DMV also exists in rats and is also glutamatergic. The second goal was to verify that this projection emanates from the C1 (PNMT-positive) neurons. To do so we located the adrenaline-synthesizing enzyme PNMT that is diagnostic of the C1 neurons and is co-expressed with TH and DβH in these neurons (Phillips et al., 2001
). The third goal was to verify, in rats, that AAV2 does not reactivate a latent glutamatergic phenotype in these catecholaminergic neurons, a possibility raised previously to explain discrepancies between electrophysiological results suggesting a contribution of glutamate to neurotransmission in adult mesolimbic dopaminergic neurons and the lack of supportive ultrastructural evidence (Stuber et al., 2010
; Moss et al., 2011
; Berube-Carriere et al., 2012
). We injected DIO eF1α EYFP AAV2 into the RVLM of 3 TH-Cre rats, which caused a high level of expression in PNMT-ir neurons (typical injection site shown in ). EYFP was also detected in a smaller number of RVLM neurons devoid of PNMT immunoreactivity suggesting some degree of ectopic Cre recombinase expression (data not illustrated). Numerous triple-labeled terminals containing EYFP, VGLUT2 and PNMT immunoreactivities were observed in each rat within the DMV (). Within this structure almost all PNMT-ir terminals were also VGLUT2-ir whether or not they were labeled with EYFP suggesting that AAV2 transduction could not have explained the presence of VGLUT2 in PNMT-ir terminals. To further verify this point, we examined the DMV of two TH-Cre rats that had not been injected with AAV2. Virtually all PNMT-ir terminals identified within the DMV were also VGLUT2-ir (). In summary, RVLM-CA neurons also innervate the DMV in rats. As in mice, the axonal projections of these neurons to the DMV contain VGLUT2 and the presence of VGLUT2 in these neurons is not an artifact caused by AAV2 transduction.
Axonal varicosities from C1 neurons in rat DMV contain PNMT and VGLUT2
ChR2-mCherry labeled terminals from RVLM-CA neurons make monosynaptic contacts with cholinergic DMV neurons
We identified 113 mCherry-ir (DAB-labeled) profiles within the DMV using tissue from 5 mice processed for EM. The DMV region examined is specified in . These profiles consisted exclusively of unmyelinated axons and nerve terminals. Immunogold-silver labeling for ChAT was observed only within perikarya and dendrites. In tissue dually labeled for mCherry and ChAT we identified 65 synaptic contacts between mCherry-ir varicosities and ChAT-ir profiles (, ). The majority of these synapses (N =48, 74%) were asymmetric () but 17 symmetric axodendritic or axosomatic synapses also were detected (26%; ). A small proportion of mCherry-positive boutons made synaptic contact onto dendrites that lacked detectable immunoreactivity for ChAT (n=18; 12 asymmetric and 6 symmetric contacts). Because ChAT immunodetection was restricted to the surface of the sections, these negative results are not clearly interpretable. They could either be false-negative results or denote that, within the confines of the DMV, RVLM-CA neurons also innervate neurons other than the parasympathetic preganglionic neurons.
RVLM-CA neurons make synapses with DMV cholinergic neurons
Synaptic contacts formed by mCherry-ir terminals and ChAT-ir neurons in DMV
Dense core vesicles consistent with catecholamine-releasing organelles were detected within the more lightly labeled mCherry-positive varicosities () but most terminals contained a level of peroxidase reaction product that was too high to reliably observe such vesicles.
Photostimulation of RVLM-CA terminals in the DMV evokes postsynaptic inward currents in DMV neurons
DMV neurons were recorded in brain slices prepared from adult DβHCre/0 mice approximately one month after injection of AAV2-DIO-ChR2-mCherry into RVLM. The dorsal vagal complex from AAV2-injected mice contained mCherry-positive fibers that were clearly visible (observed with epifluorescence using Zeiss filter set 20: excitation 546 nm, emission 575 nm). The slices used for recording did not contain mCherry-positive somata, confirming that few if any A1 noradrenergic neurons had been exposed to the AAV2. mCherry-positive somata were consistently observed in the expected ventrolateral medullary location in more rostral slices.
We believe that we recorded exclusively from DMV neurons because of their fairly obvious location in coronal slices. Moreover, in several instances when we recorded from DMV neurons visualized with Fluoro-Gold, we obtained the same results as with DMV neurons sampled without Fluoro-Gold guidance. Also, several intracellular fills labeled DMV neurons with an axon that extended mediolaterally towards an exiting vagal rootlet () as expected for parasympathetic preganglionic neurons. Finally, the membrane potential and discharge characteristics of the DMV neurons recorded in current clamp were similar to those reported previously (Browning et al., 1999
; Martinez-Pena y Valenzuela et al., 2004
Blue light (473 nm, 5mW, 1ms pulses) was delivered by placing a 200 µm thick optical fiber 300 µm above the dorsal vagal complex. The light pulses had no detectable effect in DMV neurons prepared from naïve mice (7 DMV neurons clamped at −79.4mV). In contrast, the light produced EPSCs (or EPSPs) in 71% of the DMV neurons recorded from AAV2-injected DβH-Cre mice (48 of 68 cells; 21 mice). The electrophysiological effects produced by the light were therefore ChR2-dependent. Low frequency light pulses (1ms, 0.5Hz) evoked a compound, inward postsynaptic current (PSC) in most DMV neurons clamped at a holding potential of −79.4 mV (). PSCs were elicited with a latency of 5.5 ± 0.3ms (23 neurons) from laser onset and had an amplitude of 71.3 ± 17.6pA (13 neurons). When the PSC was of small amplitude (<60 pA), occasional failures were observed (). When the evoked PSC had a larger amplitude (60–120 pA; ), it was more prominently multiphasic and a PSC after-discharge lasting nearly a full second (decay τ = 0.6s) was commonly observed ().
Photostimulation of ChR2-labeled axons produces PSCs in DMV neurons and increases their discharge rate
High frequency photostimulation (1ms, 20Hz, 5s), produced a very large increase in PSC frequency with a concurrent inward shift in the holding current (). Unlike with the single pulse paradigm, the PSCs elicited by train stimulation occurred at random relative to the laser pulses (: stim). Accordingly, during train stimulation, stimulus-triggered waveform averages were flat (). Train stimulation almost always produced a strong after-discharge of PSCs that took greater than 30 seconds to return to the pre-stimulus (baseline) frequency (: afterdischarge; ). PSCs were binned in 10s intervals following the end of the photostimulus and between groups differences were determined by non-parametric Friedman’s analysis (F=87.4; P<0.0001) followed by Dunn’s post hoc test for multiple comparisons (see ). The decay in PSC frequency following a train of stimuli could be modeled with two exponentials (τfast=2.3 ± 0.4 s; τslow=48 ± 18 s, N=16 neurons).
We also recorded from DMV neurons in whole-cell current-clamp mode to determine whether the excitatory input from RVLM-CA neurons was strong enough to elicit action potentials. On average, DMV neurons had a resting membrane potential of −63 ± 1.5mV (N = 7) and a baseline-firing rate of 0.8 ± 0.2Hz. In every recorded cell (N = 8), photostimulation of RVLM-CA terminals using a 5-second train (1ms, 20Hz) of laser light bursts produced a massive increase in EPSP frequency and markedly increased the resting discharge rate of the recorded neuron. The DMV neuron illustrated in was kept just below firing threshold by injection of bias current (−10 pA) and the first differential of the original voltage recording (dV/dt) is shown as a means of better distinguishing the occurrence of postsynaptic potentials. In the 7 cells in which no bias current was applied, discharge rate was increased from 0.8 ± 0.3 spikes/s to 3.7 ± 0.8 spikes/s (p<0.01, paired t-test).
Photostimulation of ChR2-expressing RVLM-CA terminals elicits excitatory PSCs exclusively
Photostimulation (5s trains, 1 ms, 5 mW) was performed while the DMV neurons were clamped at two discrete holding potentials to differentiate excitatory, cationic currents (inward; Vm = −79.4 mV) from inhibitory, anionic currents (outward; Vm = −9.4mV; representative examples in ). At Vm of −79.4 mV the photostimulus train produced a barrage of inward, short-lasting EPSCs (decay τ = 6.5 ± 0.3ms; amplitude = 12.7 ± 1.8pA; N = 8; ). When the neuron was clamped at Vm= −9.4 mV, longer-lasting outward IPSCs (decay τ = 16.5 ± 1.1ms; amplitude = 20.7 ± 1.5pA; n=8) were typically observed during the resting period, but the frequency of these events was unaffected by photostimulation (). At rest the recorded DMV neurons received an approximately equal frequency of EPSCs and IPSCs (). Two-way repeated-measures ANOVA followed by Bonferroni post hoc test for multiple comparisons indicated that only EPSC frequency was altered during photostimulation. Influence of photostimulation; F(2,32)=54.43, P<0.0001. Influence of PSC (EPSC vs. IPSC); F(1,16)=8.56, P<0.01. Interaction of photostimulation and PSC; F(2,32)=51.2, P<0.0001.
Photostimulation of ChR2-expressing axons produces exclusively EPSCs in DMV neurons
Thus, based on this sample of 8 neurons, photostimulation of the axons of RVLM-CA neurons does not elicit IPSCs in DMV neurons. The absence of detectable mRNA transcripts for glycine transporter 2 or glutamic acid decarboxylase in RVLM-CA neurons in rats agrees with these findings (Stornetta and Guyenet, 1999
; Stornetta et al., 2004
The EPSCs evoked in DMV neurons by stimulating RVLM-CA terminals are glutamatergic
The strong increase in EPSC frequency and associated inward shift in membrane current elicited by trains of photostimuli (1 ms, 20 Hz, 5s) in DMV neurons clamped at −79.4mV was unchanged by the addition of bicuculline and strychnine to block GABA and glycine–mediated currents, leaving the total integrated current (area under the curve, AUC) during the stimulus period unchanged (). However, addition of the NMDA/AMPA antagonists AP5/CNQX produced near complete blockade of spontaneous EPSCs and EPSCs elicited during the photostimulation (). Group data were analyzed with one-way ANOVA for repeated measures, (F(2,6)=7.7; P<0.05) followed by Student-Newman-Keuls (SNK) post hoc test for multiple comparisons. The AP5/CNQX mixture also greatly attenuated the peak amplitude (93.1 ± 2.6%) and integrated AUC (91.1 ± 2.4%) of the averaged compound EPSC evoked by single pulse photostimulation (1ms, 0.5Hz; N = 4; not illustrated). The broad spectrum excitatory amino acid antagonist kynurenic acid (1 mM) reversibly reduced background EPSC activity and also markedly attenuated the inward current generated by the trains of photostimuli (1 ms 20 Hz, 5s; 82.3 ± 3.9% attenuation; P<0.01; ). Like AP5/CNQX, kynurenic acid also significantly reduced the peak amplitude and integrated AUC of the EPSCs elicited by low frequency stimulation (respectively 66.2 ± 4.4% and 72.7 ± 4.1% attenuation; P<0.01; N = 9; not illustrated). Group data for effects of kynurenic acid were analyzed by one-way ANOVA for repeated measures, (F(2,22)=7.8; P<0.01) followed by SNK post hoc test for multiple comparisons.
EPSCs evoked in DMV neurons are glutamatergic
Glutamatergic input from RVLM-CA neurons to DMV neurons is monosynaptic
To determine if the excitatory glutamatergic input from RVLM-CA neurons to the DMV was mono- or poly-synaptic, we first blocked voltage-gated sodium (Nav
) channels with 1µM tetrodotoxin (TTX), then we applied 100 µM 4-aminopyridine (4-AP) to block the Kv
channels that are critical for the repolarization of the axon (Shu et al., 2007
). This protocol eliminates action potential-dependent, therefore potentially polysynaptic events, but enhances the direct depolarization of ChR2-positive terminals and the local release of neurotransmitter during photostimulation (Petreanu et al., 2009
). As found by the latter authors, the compound EPSC evoked by single-pulse stimulation (1ms, 0.5Hz) was virtually eliminated by TTX (). After co-application of TTX and 4-AP, a slower, ChR2-dependent EPSC that relied on glutamate transmission was reinstated (). Group data were analyzed by the non-parametric Kruskal-Wallis test (H=14.3, P<0.01) followed by Dunn’s post hoc test for multiple comparisons. Use of a 5-second train of stimuli (1 ms, 20 Hz) produced similar results (). Application of TTX alone significantly blunted the overall response to train stimulation. The increase in evoked EPSCs was delayed and only at the very end of the 5-second stimulus period did a small increase in EPSC frequency become apparent. This suggested that TTX imposed a significant slowing of the terminal depolarization during the photostimulus, requiring seconds to reach a “threshold” for Ca2+
entry and vesicular docking. Co-application of TTX and 4-AP restored the response towards the control value, particularly the rapid increase in EPSCs occurring early in the stimulus train (). This action potential-independent transmission relied on ionotropic glutamate receptors as blockade with NMDA/AMPA antagonists eliminated the effect of photostimulation (). Group data were analyzed by one-way ANOVA, F(3.21)=17.8, P<0.0001) followed by SNK post hoc test for multiple comparisons. The number of repeats per group is unequal due to loss of recorded neurons at different points along the experiments. Significance of comparisons: CNQX/AP5 vs. control and TTX vs. control, p<0.001. 4AP vs. control, p<0.01. CNQX/AP5 vs. TTX/4AP and TTX vs. 4AP, p<0.05. CNQX/AP5 vs. TTX, n.s.
Photostimulation of ChR2-expressing fibers activate DMV neurons after action potential blockade