Cerebellar modulation of synaptic input to freezing-related neurons in the periaqueductal gray
- Department of Neurobiology, Northwestern University, United States
Figures
Figure 1 with 1 supplement
Anatomical tracing and identification of Chx10-positive vlPAG neurons.
(A) Left, Schematic of a parasagittal section of the cerebellum showing injection site for anterograde tracing from mCbN. Middle, Confocal image of the mCbN after injection of AAV-hSyn-ChR2-eYFP …
Figure 1—figure supplement 1
Low magnification image of mCbN labeled axons in vlPAG.
(A) Schematic of the approximate rostro-caudal level of the highest density of mCbN labeled fibers in the vlPAG. Box denotes boundaries of image in panel (B). Abbreviations: SuG: superficial grey …
Figure 2
Freezing evoked by activation of Chx10 neurons in vivo.
(A) Action potentials in a Chx10-ChR2 expressing neuron in the vlPAG during a train (100 stimuli, 50 Hz, 10 ms) of light. (B) Spike raster of 10 consecutive sweeps during stimulation of the cell in …
Figure 3 with 1 supplement
Intrinsic and synaptic properties of Chx10 neurons in PAG slices.
(A) Spontaneous action potentials from a Chx10 neuron. (B) Population data for spontaneous firing rates of Chx10 neurons. (C) Single spontaneous action potential of a Chx10 neuron. (D) Phase-plane …
Figure 3—figure supplement 1
Comparison of Chx10 and GAD2 intrinsic properties.
(A) Spontaneous action potentials in a GAD2+ (green) and a Chx10 (black) neuron. (B) Population data for spontaneous firing rates of GAD2 and Chx10 neurons. Open symbols, individual cells, solid …
Figure 4 with 1 supplement
Intrinsic and synaptic properties of mCbN neurons in cerebellar slices.
(A) Spontaneous action potentials recorded from mCbN neurons from males (blue) and females (red). (B) Population data of spontaneous firing rates separated by sex. Open symbols, individual cells, sol…
Figure 4—figure supplement 1
Comparison of mCbN and iCbN intrinsic and synaptic properties in males and females.
(A, B) Spontaneous action potentials of iCbN (left) and mCbN (right) neurons from females (A) and males (B). (C) Mean and SEM of firing rates of iCbN and mCbN neurons, pooled for both sexes. Asterisk…
Figure 5
Direct and modulated PSCs in Chx10 cells evoked by mCbN optogenetic stimulation.
(A) EPSCs evoked in a Chx10 cell by optogenetic stimulation of mCbN afferents. (B) Percentage of tested cells in which responses could be elicited (grey bars) and population data for EPSC amplitudes …
Figure 6
EPSCs in TH neurons evoked by mCbN activation.
(A) Distribution of TH neurons in the vlPAG. (B) Optogenetically evoked EPSCs in a TH neuron. (C) Population data for the first EPSC (left) and PPR (right) evoked as in B. Open symbols, individual …
Figure 7
Modulation of PSCs in unlabeled vlPAG cells evoked by TH neuron stimulation.
(A) Top, protocol for stimulating electrically (upper line) and optogenetically (lower line). Bottom, IPSCs without (black) and with (color) mCbN stimulation. (B) Population data for percent change …
Figure 8
PSC modulation in Chx10 cells by D2 but not D1 receptor activation.
(A) Left, Electrically evoked IPSCs before (black) and during (color) bath application of the D2-receptor agonist quinpirole (25 μM). Right, As at left, but with D1 receptors activated by dopamine …
Figure 9
Blockade of mCbN-dependent modulation of Chx10 neuron PSCs by D2 receptor antagonists.
(A) Top, protocol for stimulating electrically (upper line) and optogenetically (lower line). Bottom, IPSCs without (black) and with (color) mCbN stimulation in the presence of sulpiride. (B) …
Figure 10
Cerebellar influence on the vlPAG freezing-related microcircuit.
(A) mCbN afferents (blue) excite Chx10 (red), GAD2 (green) and TH (purple) neurons in the vlPAG, with the strongest functional connection occurring between mCbN neurons and TH neurons. Chx10 neurons …
Videos
Video 1
Optogenetic stimulation of Chx10 neurons in vlPAG elicits freezing.
Responses evoked by optogenetic activation of Chx10 neurons in the vlPAG (10 ms, 50 Hz, 5 s) is indicated by ‘Light On.’.
Tables
Table 1
Intrinsic properties of Chx10+ vlPAG neurons.
| Parameter | All Cells (n=26) | Males (n=15) | Females (n=11) | p value |
|---|---|---|---|---|
| Intrinsic Properties | ||||
| Membrane Resistance (MΩ) | 584.1 ± 47.3 | 648.6 ± 62.2 | 496.0 ± 66.9 | 0.1 |
| Capacitance (pF) | 24.2 ± 1.3 | 23.4 ± 1.5 | 25.2 ± 2.5 | 0.6 |
| Interspike Vm (mV)* | -48.7 ± 1.0 | -50.6 ± 1.0 | -45.9 ± 1.6 | 0.02 |
| Action Potential Properties | ||||
| Spontaneous Rate (spikes/s) | 5.8 ± 1.2 | 4.5 ± 1.4 | 7.6 ± 1.9 | 0.2 |
| Maximum dV/dt (V/s) | 155.2 ± 11.4 | 153.9 ± 13.5 | 157.0 ± 20.5 | 0.9 |
| Estimated Peak INa (nA) | 4.0 ± 0.5 | 3.7 ± 0.5 | 4.3 ± 0.9 | 0.6 |
| Minimum dV/dt (V/s) | -63.9 ± 5.0 | -55.1 ± 4.0 | -76.0 ± 9.5 | 0.06 |
| Estimated Peak IK (nA) | 1.6 ± 0.2 | 1.3 ± 0.1 | -2.1 ± 0.4 | 0.06 |
| Halfwidth (ms) | 1.0 ± 0.1 | 1.1 ± 0.07 | 0.9 ± 0.1 | 0.06 |
| Amplitude (mV) | 55.9 ± 2.2 | 57.3 ± 2.7 | 54.1 ± 3.5 | 0.5 |
| Threshold (mV) | -31.6 ± 0.8 | -32.5 ± 1.0 | -30.4 ± 1.4 | 0.2 |
| Rheobase at -70 mV (pA) | 9.8 ± 0.8 | 9.6 ± 1.0 | 10.0 ± 1.4 | 0.8 |
-
* indicates significant difference between males and females.
Table 2
Intrinsic properties of medial cerebellar nucleus neurons.
| Parameter | All Cells (n=28) | Males (n=15) | Females (n=13) | p value |
|---|---|---|---|---|
| Intrinsic Properties | ||||
| Membrane Resistance (MΩ) | 77.5 ± 12.8 | 62.2 ± 8.1 | 95.5 ± 17.0 | 0.08 |
| Capacitance (pF) | 68.6 ± 5.5 | 79.7 ± 12.6 | 55.2 ± 5.8 | 0.09 |
| Action Potential Properties | ||||
| Spontaneous Rate (spike/s)* | 122.8 ± 6.6 | 137.4 ± 8.8 | 105.9 ± 9.0 | 0.01 |
| Maximum dV/dt (V/s) | 294.3 ± 19.0 | 270.6 ± 16.6 | 321.8 ± 34.6 | 0.2 |
| Estimated Peak INa (nA) | 19.4 ± 2.0 | 20.8 ± 2.9 | 17.9 ± 2.8 | 0.5 |
| Minimum dV/dt (V/s) | -270.7 ± 20.1 | -242.1 ± 19.4 | -303.4 ± 38.4 | 0.2 |
| Estimated Peak IK (nA) | 17.8 ± 1.8 | 18.4 ± 2.4 | 17.2 ± 3.0 | 0.7 |
| Halfwidth (ms) | 0.27 ± 0.02 | 0.27 ± 0.2 | 0.27 ± 0.4 | 0.9 |
| Ampliutde (mV) | 53.2 ± 1.5 | 51.7 ± 1.7 | 54.9 ± 2.8 | 0.3 |
| Threshold (mV) | -42.3 ± 0.7 | -41.7 ± 1.0 | -43.0 ± 1.1 | 0.4 |
-
* indicates significant difference between males and females.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional Information |
|---|---|---|---|---|
| Strain, strain background Mus musculus | Chx10-cre | Obtained from Jessell Laboratory (Crone et al., 2008) | ||
| Strain, strain background Mus musculus | tdTomato (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J) | Jackson Laboratories | Stock: 007914 | |
| Strain, strain background Mus musculus | ChR2-EYFP (B6.Cg-Gt(ROSA)26 Sortm32(CAG-COP4*H134R/EYFP)Hze/J) | Jackson Laboratories | Stock: 024109 | |
| Strain, strain background Mus musculus | GAD2-cre (B6N.Cg-Gad2tm2(cre)Zjh/J) | Jackson Laboratories | Stock:019022 | |
| Strain, strain background Mus musculus | TH-cre (B6.Cg-7630403G23 RikTg(Th-cre)1Tmd/J) | Jackson Laboratories | Stock: 008601 | |
| Strain, strain background Mus musculus | L7-Cre (B6.Cg-Tg(Pcp2-cre)3555Jdhu/J) | Jackson Laboratories | Stock: 010536 | |
| Recombinant DNA reagent | AAV2-EF1α-DIO-hChR2(H134R)-eYFP | UNC Viral Vector Core | ||
| Recombinant DNA reagent | AAV2-hSyn-hChR2 (H134R)-eYFP-WPRE-PA | UNC Viral Vector Core | ||
| Recombinant DNA reagent | AAVdj-hSyn-hChR2(E123A)-eYFP-WPRE | Stanford Viral Vector Core | ||
| Recombinant DNA reagent | AAV2-EF1α-DIO-eYFP | UNC Viral Vector Core | ||
| Chemical compound, drug | Cholera Toxin Subunit B (Recombinant), Alexa Fluor 488 Conjugate | ThermoFisher Scientific | Cat. No. C22841 | |
| Chemical compound, drug | Lumafluor red retrobeads | LumaFluor | Red Retrobeads IX | |
| Chemical compound, drug | DNQX | Tocris | Cat. No. 0189 | |
| Chemical compound, drug | CPP | Tocris | Cat. No. 0247 | |
| Chemical compound, drug | CPCCOEt | Tocris | Cat. No. 1028 | |
| Chemical compound, drug | Sulpiride | Tocris | Cat. No. 0894 | |
| Chemical compound, drug | SCH 23390 | Tocris | Cat. No. 0925 | |
| Chemical compound, drug | Prazosin | Tocris | Cat. No. 0623 | |
| Chemical compound, drug | Sotalol | Tocris | Cat. No. 0952 | |
| Chemical compound, drug | Quinpirole | Tocris | Cat. No. 1061 | |
| Chemical compound, drug | Dopamine | Tocris | Cat. No. 3548 | |
| Chemical compound, drug | Isoproterenol | Tocris | Cat. No 17.47 | |
| Software, algorithm | FreezeFrame | Actimetrics (www.actimetrics.com) |
