dreadd ligand clozapine n oxide  (Tocris)

Journal: eLife

Article Title: Multiple functions of cerebello-thalamic neurons in learning and offline consolidation of a motor skill in mice

doi: 10.7554/eLife.102813

Figure Lengend Snippet: ( a ) Scheme of the implantation and injection. ( b ) Coronal section of the cerebellum showing hM4Di-DREADD expression in the three CN. ( c ) Representative confocal image showing hM4Di-DREADD expression on neuronal membranes. ( d ) Electrode placement close to cells expressing hM4Di-DREADD (red: lesion site, yellow: electrode track). ( e ) Examples of high-pass filtered traces of CN recordings before and after CNO injection. ( f ) Examples of spike shapes obtained from spike sorting in CN (line + shading indicates mean +/- SD). ( g ) Boxplots displaying the percentage of modulation of CN neurons average firing rate induced by CNO or SAL injections during an open field session. CN firing rate was reduced after 1 mg/kg of CNO injection in DREADD-injected mice compared to other groups (Wilcoxon test* p<0.05, ***p<0.001. Boxes represent quartiles and whiskers correspond to range; points are singled as outliers if they deviate more than 1.5 x interquartile range from the nearest quartile). ( h ) Latency to fall during fixed speed rotarod (5, 10, 15, 20 r.p.m.) for all experimental groups. One way repeated-measure ANOVA on averaged values for all the speed steps in each experimental group followed by Tukey Post hoc pairwise comparison (p>0.05 in all cases). ( i ) Impact of daily injections of CNO before trial 1 on accelerating rotarod performance. Summary of the performance for each trial/day (repeated measure ANOVA Group effect *p<0.05, ***p<0.001; # p<0.05, ### Pp <0.001 Tukey pairwise test last trial of each day vs first trial of next day).( j ) Impact of daily injections of CNO after the task (30 min after trial 7). All treatment is switched to saline in the Late phase. Same presentation as in ( i ). Data in h,i,j are presented as mean ± S.E.M. n indicates the number of mice.

Article Snippet: The DREADD ligand Clozapine-N-Oxide (CNO, TOCRIS, Bristol, UK) was dissolved in SAL (0.9% sodium chloride) and injected intraperitoneally at 1 mg/kg.

Techniques: Injection, Expressing, Comparison, Saline

Journal: eLife

Article Title: Multiple functions of cerebello-thalamic neurons in learning and offline consolidation of a motor skill in mice

doi: 10.7554/eLife.102813

Figure Lengend Snippet: DAPI-positive neurons expressing hM4Di-DREADD-mCherry in cerebellar nuclei.

Article Snippet: The DREADD ligand Clozapine-N-Oxide (CNO, TOCRIS, Bristol, UK) was dissolved in SAL (0.9% sodium chloride) and injected intraperitoneally at 1 mg/kg.

Techniques: Expressing

Journal: eLife

Article Title: Multiple functions of cerebello-thalamic neurons in learning and offline consolidation of a motor skill in mice

doi: 10.7554/eLife.102813

Figure Lengend Snippet: ( a ) Footprint patterns were quantitatively assessed for three parameters as shown on representative footprint patterns for all experimental groups. Three parameters are represented graphically: linear movement (bottom left), sigma (bottom middle), and alternation coefficient (bottom right). ( b ) Tests of strength and coordination. Grid test: latency reflecting the time before falling from the grid. 30 s of cut-off was established as the maximum latency (dotted line on figure). Horizontal bar: latency to cross the horizontal bar (balance beam test) for all experimental groups. Vertical pole: latency to reach home cage in vertical pole test for all experimental groups. ( c ) Locomotor activity (Velocity) in DREADD and non-DREADD (Sham) injected mice after CNO or SAL injection during open-field sessions before (OF1) and after (OF2) rotarod for experimental days 1, 4, and 7. n indicates the number of mice. Boxes in a,b represent quartiles and whiskers correspond to range; points are singled as outliers if they deviate more than 1.5 x interquartile range from the nearest quartile. Data in c are represented as mean ± S.E.M. No post hoc significant difference was found for the data presented in this figure.

Article Snippet: The DREADD ligand Clozapine-N-Oxide (CNO, TOCRIS, Bristol, UK) was dissolved in SAL (0.9% sodium chloride) and injected intraperitoneally at 1 mg/kg.

Techniques: Activity Assay, Injection

Journal: eLife

Article Title: Multiple functions of cerebello-thalamic neurons in learning and offline consolidation of a motor skill in mice

doi: 10.7554/eLife.102813

Figure Lengend Snippet: ( a ) Example of evolution of latencies to fall for a mouse during the accelerating rotarod protocol. Linear regressions estimated values of trials 1 and 7 during each day are shown using green hollow dots, within-day learning (green arrows), overnight change (red arrows), and day + night learning (brown arrows). ( b ) Evolution of estimated daily learning, overnight change, and consolidated learning shows learning mostly in days 1–4 (Early Phase). Plots represent mean ± S.E.M. ( c ) Within-day learning vs daily initial performances (top) and within-day learning vs consolidated learning (bottom) in the Early phase. Scatterplot of performance from all control mice; the ellipse contains 50% of a bivariate normal distribution fitted to the values and the dot indicates the center of the distribution. Deming linear regression outcomes are represented with 95% confidence interval in shaded color. ( d ) Same as panel (c) with superimposition of controls (black) and mice expressing DREADD in DCN and CNO during the task (orange); only ellipses and regression line are included for clarity of the graph (**p<0.01 ***p<0.001 Wilcoxon test for difference between groups of residuals, i.e. signed distance of performances to Deming regression line of control mice). ( e ) Same as panel ( d ) for CNO administered after the task.

Article Snippet: The DREADD ligand Clozapine-N-Oxide (CNO, TOCRIS, Bristol, UK) was dissolved in SAL (0.9% sodium chloride) and injected intraperitoneally at 1 mg/kg.

Techniques: Control, Expressing

Journal: eLife

Article Title: Multiple functions of cerebello-thalamic neurons in learning and offline consolidation of a motor skill in mice

doi: 10.7554/eLife.102813

Figure Lengend Snippet: ( a ) Scheme of the combined viral injections targeting the CN->CL neurons using a retrograde virus expressing the Cre in the thalamus and a virus inducing Cre-dependent expression of inhibitory DREADD in the CN. Top : schematic of the viral injections. Bottom : GFP fluorescence revealing the site of injection of the CAV viruses. ( b ) Comparison of the effect of daily injections before trial 1 of CNO (orange) or Saline (black) in mice described in panel ( a ) (Data represents mean ± S.E.M, n indicates the number of mice; *p<0.05, **p<0.01, ***p<0.001; repeated measure ANOVA Group effect). ( c ) Same as ( b ) for daily CNO injections 30 min after trial 7 during the Early Phase. ( d ) Scheme of combined viral injections targeting the CN->VAL neurons. ( e,f ) Same as ( b,c ) for CNO-injected (blue) and control (black) mice obtained as described in panel d. ( g ) Same as for the Saline-treated groups of panels b–f. ( h–k ) Same as for the different groups receiving injections CNO during or after the task compared to Saline-treated mice. Same color coding as panels b, c and e, f. (**p<0.01 ***p<0.001 Wilcoxon test for difference between groups of residuals i.e. signed distance of performances to Deming regression line of control mice). CN: cerebellar nuclei, VAL: ventral anterior lateral thalamus, CL: central lateral thalamus.

Article Snippet: The DREADD ligand Clozapine-N-Oxide (CNO, TOCRIS, Bristol, UK) was dissolved in SAL (0.9% sodium chloride) and injected intraperitoneally at 1 mg/kg.

Techniques: Virus, Expressing, Fluorescence, Injection, Comparison, Saline, Control

Journal: eLife

Article Title: Multiple functions of cerebello-thalamic neurons in learning and offline consolidation of a motor skill in mice

doi: 10.7554/eLife.102813

Figure Lengend Snippet: ( a ) Footprint patterns were quantitatively assessed for three parameters as shown on representative footprint patterns (top) for all experimental groups. Three parameters are represented graphically: linear movement (bottom left), sigma (bottom middle), and alternation coefficient (bottom right). ( b ) Latency reflecting the time before falling from the grid. 30 seconds of cut-off was established as the maximum latency (dotted line on figure). ( c ) Latency to cross the horizontal bar (balance beam test) for all experimental groups. ( d ) Latency to reach home cage in vertical pole test for all experimental groups. ( e ) Locomotor activity (Velocity) in DREADD-injected mice after CNO or SAL injection during open-fields sessions before (OF1) and after (OF2) rotarod for days 1, 4, and 7 (**p<0.01 paired t-test OF1 vs OF2). ( f ) Latency to fall during fixed speed rotarod (5, 10, 15, 20 r.p.m.) for all experimental groups. One-way repeated measures ANOVA was performed on averaged values for each speed step in each experimental group followed by a Tukey post hoc pairwise comparison (*p<0.05, **p<0.01 for CN->VAL group CNO vs SAL). CN, cerebellar nuclei, CL, centrolateral thalamus; VAL, ventral anterior lateral thalamus. n indicates the number of mice. Boxes represent quartiles and whiskers correspond to range; points are singled out as outliers if they deviate more than 1.5 x interquartile range from the nearest quartile. Plots in e,f represent mean ± S.E.M.

Article Snippet: The DREADD ligand Clozapine-N-Oxide (CNO, TOCRIS, Bristol, UK) was dissolved in SAL (0.9% sodium chloride) and injected intraperitoneally at 1 mg/kg.

Techniques: Activity Assay, Injection, Comparison

Journal: eLife

Article Title: Multiple functions of cerebello-thalamic neurons in learning and offline consolidation of a motor skill in mice

doi: 10.7554/eLife.102813

Figure Lengend Snippet: ( a ) Performance of mice with DREADD expression in CL-projecting CN neurons, which learned the task under respectively CNO and Saline treatment during 7 days and then received, respectively, Saline and CNO treatment during a Reversal phase (*: p<0.05 repeated-measure ANOVA group effect; Data represented as mean ± S.E.M, n indicates the number of mice). ( b ) Change induced by treatment switch on skill levels for the first trial (daily start) and last trial (daily end); values are estimated as in , and start and end values for days 8 and 9 are averaged for each animal; *p<0.05, **p<0.01, ***p<0.001 paired Wilcoxon test. Boxes represent quartiles and whiskers correspond to range; points are singled as outliers if they deviate more than 1.5 x interquartile range from the nearest quartile. ( c,d ) Same as panels a and b for mice with DREADD expression in VAL-projecting CN neurons . CN: cerebellar nuclei, VAL: ventral anterior lateral thalamus, CL: central lateral thalamus.

Article Snippet: The DREADD ligand Clozapine-N-Oxide (CNO, TOCRIS, Bristol, UK) was dissolved in SAL (0.9% sodium chloride) and injected intraperitoneally at 1 mg/kg.

Techniques: Expressing, Saline

Journal: bioRxiv

Article Title: The importance of M1 muscarinic receptor phosphorylation in learning and memory

doi: 10.64898/2026.03.23.713145

Figure Lengend Snippet: M1-WT-HA (n=11-14) (A) , M1-KO (n=13-16) (B) or M1-PD-HA (n=11-14) (C) mice were treated with 1.5 mg/kg scopolamine +/-10 mg/kg VU846 30 mins prior to training in a fear conditioning paradigm. Mice were then returned to the same environment 24 hrs later for contextual memory retrieval whereby a reduced level of freezing indicates impaired memory. The response in vehicle treated animals across strains was also compared (D) . 2-way ANOVA with Tukey’s post-hoc correction for multiple comparisons, *p<0.05, ***p<0.001, ****p<0.0001, ns=not significant. (E ) Representative images of CA1 hippocampal region of HA-tagged M1-WT, M1-DREADD, M1-PD and M1-DREADD-PD stained with HA antibody demonstrating location of the receptor (green). Images at 40X magnification, scale bar = 50 µm

Article Snippet: The drugs utilised in this study were the pan-muscarinic antagonist, to induce a learning and memory (LM) deficit, scopolamine hydrobromide (Sigma) at 0.5-3 mg/kg doses; the M1 positive allosteric modulator (PAM) VU0486846 (VU846) at 10-100 mg/kg; and the M1-DREADD activating ligand clozapine-n-oxide (CNO) (Tocris) at 0.3 mg/kg.

Techniques: Staining

Journal: bioRxiv

Article Title: The importance of M1 muscarinic receptor phosphorylation in learning and memory

doi: 10.64898/2026.03.23.713145

Figure Lengend Snippet: (A) Male and female M1-WT-HA (n=11) and M1-PD (n=12) mice were treated with the M1-PAM VU846 30 mins prior to fear conditioning and returned to the chamber 24 hrs later for contextual memory retrieval. 2-way ANOVA ****p<0.0001 effect of strain, no significant effect of drug overall p=0.12, Bonferroni’s post-hoc correction for multiple comparisons between vehicle and VU846 for each strain, ns= not significant. (B & C) Male M1-DREADD mice were treated with 0.3 mg/kg of clozapine-N-oxide (CNO), an orthosteric agonist of the DREADD receptor, or vehicle (n=4-5 per group) 30 mins prior to training in a fear conditioning paradigm and then returned to the same environment 24 hrs later for contextual memory retrieval (B) or presentation of the tone in an altered environment 48 hrs later for cued memory retrieval (C) whereby a reduced level of freezing indicates impaired memory. (D) WT mice (n=7-9 per group) were also treated with 0.3 mg/kg CNO in a separate experiment demonstrating no effect of CNO. 2-way ANOVA with Dunnett’s post-hoc correction for multiple comparisons, ****p<0.0001, all other comparisons p>0.05.

Article Snippet: The drugs utilised in this study were the pan-muscarinic antagonist, to induce a learning and memory (LM) deficit, scopolamine hydrobromide (Sigma) at 0.5-3 mg/kg doses; the M1 positive allosteric modulator (PAM) VU0486846 (VU846) at 10-100 mg/kg; and the M1-DREADD activating ligand clozapine-n-oxide (CNO) (Tocris) at 0.3 mg/kg.

Techniques:

Journal: bioRxiv

Article Title: The importance of M1 muscarinic receptor phosphorylation in learning and memory

doi: 10.64898/2026.03.23.713145

Figure Lengend Snippet: Male M1-DREADD mice were treated with 0.01 - 0.3 mg/kg of clozapine-N-oxide (CNO), an orthosteric agonist of the DREADD receptor, or vehicle (n=5-9 per group) 30 mins prior to training in a fear conditioning paradigm and then returned to the same environment 24 hrs later for contextual memory retrieval. Data presented as freezing level normalised to the highest freezing response. One way ANOVA with Bonferroni’s post hoc correction for multiple comparisons to vehicle treatment group *p<0.05. All other comparisons not significant.

Article Snippet: The drugs utilised in this study were the pan-muscarinic antagonist, to induce a learning and memory (LM) deficit, scopolamine hydrobromide (Sigma) at 0.5-3 mg/kg doses; the M1 positive allosteric modulator (PAM) VU0486846 (VU846) at 10-100 mg/kg; and the M1-DREADD activating ligand clozapine-n-oxide (CNO) (Tocris) at 0.3 mg/kg.

Techniques:

Journal: Cell reports

Article Title: Vasopressin and oxytocin excite BNST neurons via oxytocin receptors, which reduce anxious arousal

doi: 10.1016/j.celrep.2025.115768

Figure Lengend Snippet: (A) In type III neurons, AVP with the V1aR/OTR antagonist did not change the I/O relationship ( p = 0.2742, n = 8). (B) OT induced a leftward shift in the I/O relationship ( p = 0.0012, n = 8). (C) OTR agonist, TGOT, shifted the I/O relationship ( p = 0.0125, n = 8). (C′) Type III neuron responses to a 90-pA pulse during TGOT. (D) V1aR agonist, FE201874 , induced an excitatory effect on the I/O relationship ( p = 0.0071, n = 10). (D′) Type III neuron responses to 90-pA pulse during FE201874 . (E) V1bR agonist, d[Cha4]-AVP, did not affect the I/O relationship ( p = 0.3535, n = 8). (F) Nissl (left) and anatomical annotations (right) from the Allen-Brain-Atlas. High somatodendritic OTR-mCherry expression in the BNST DL of OTR-Cre rats injected with AAV-DIO-DREADDs-mCherry. (G and H) Responses of fluorescent-mCherry-OTR (G) type I and (H) type III/OTR neuron to hyperpolarizing and depolarizing current-pulses. (I) In type III/OTR-mCherry neurons, AVP significantly increased SSF ( p = 0.0003, n = 12). (I′) Type III/OTR-mCherry neuron responses to 130-pA pulse during AVP. (J) The majority of OTR-BNST DL -mCherry neurons were classified as type III (35/45). (K) The majority of CRF-BNST DL -mCherry neurons (21/26) were also type III. (L) In type III/CRF neurons, AVP increased SSF ( p = 0.0432, n = 7). (L′) Type III/CRF neuron responses to 100-pA current pulse during AVP. (M) Nissl (left) and anatomical annotations (right) from the Allen-Brain-Atlas. High CRF-mCherry expression in the BNST DL after CRF-Cre rats were injected with AAV-DIO-DREADDs-mCherry. (N) Responses of type III/CRF-mCherry neuron to hyperpolarizing and depolarizing currents. (O) In type III/CRF neurons, TGOT significantly increased SSF ( p = 0.0211, n = 7), with trends at 140 pA ( p = 0.0697) and 150 pA ( p = 0.0780, vs. pre). (O′) Type III/CRF-mCherry neuron responses to 150-pA pulse during TGOT. Electrophysiological data are presented as mean ± standard error of mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. pre. ac, anterior commissure; BNST AM , anteromedial BNST; BNST OV , oval nucleus of the BNST; CP, caudate putamen; IC, internal capsulae; LV, lateral ventricle.

Article Snippet: DREADDs ligand (Clozapine-N-oxide, CNO) , Tocris, Bio-Techne Corporation , 4936.

Techniques: Expressing, Injection

Journal: Cell reports

Article Title: Vasopressin and oxytocin excite BNST neurons via oxytocin receptors, which reduce anxious arousal

doi: 10.1016/j.celrep.2025.115768

Figure Lengend Snippet: (A) Nissl (left) and anatomical annotations (right) from the Allen-Brain-Atlas. Male OTR-Cre rats injected with AAV-DIO-DREADDs-Gi-mCherry into the BNST DL were treated with CNO or saline before fear-conditioning in the FPS and again, before the EPM. (B) In vitro patch-clamp electrophysiology in brain slices from the OTR-Cre rats shows that CNO reduces the firing frequency of OTR-BNST-mCherry neurons. (C) FPS apparatus with Plexiglass enclosure and startle detection. (D–D″) During the first FPS-recall, there was a trial effect of noise-only vs. light and noise ( p < 0.0001), and post-shock vs. noise-only conditions ( p = 0.0109), but no treatment effects (D). No significant differences were found in the percentage change of cued fear (D′) or non-cued fear (D″). (E–E″) During the second FPS, there was a trial effect between the noise-only and light and noise conditions ( p < 0.0001), and a treatment effect ( p = 0.0477). There was a trial effect between the post-shock and noise-only conditions ( p = 0.0024), a treatment effect ( p = 0.0307), and an interaction ( p = 0.0430) (E). There were no differences in the percentage change of cued fear (E′), but the percentage change of non-cued fear (anxious arousal) was significantly higher in CNO vs. saline controls ( p = 0.0077) (E″). (F–K) Representative traces of locomotor activity during the EPM (F). Rats treated with CNO spent less time in the open-arms (G), spent more time in the closed-arms (H), and tended to spend less time in the center (I) vs. saline controls. No significant differences were found between groups in the number of closed-arms entries (J). CNO-treated rats spent significantly more time freezing in the closed-arms vs. saline-treated rats ( p = 0.0344) (K). * p < 0.05, ** p < 0.01.

Article Snippet: DREADDs ligand (Clozapine-N-oxide, CNO) , Tocris, Bio-Techne Corporation , 4936.

Techniques: Injection, Saline, In Vitro, Patch Clamp, Activity Assay