Cannabinoid overrides triggers of GABAergic plasticity in vestibular circuits and distorts the development of navigation
School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, P.R. China
State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, P.R. China
Neuroscience Research Centre, The University of Hong Kong, Pokfulam, Hong Kong SAR, P.R. China
Beijing Advanced Innovation Center for Big Data-based Precision Medicine, School of Engineering and Medicine, Beihang University, Beijing, P.R. China
Summary
Early life exposure to cannabis can result in long-lasting deficits in spatial navigation. We ask if the development of this behavior is subject to early life activity of type I cannabinoid receptor (CB1R) in the vestibular nucleus. In rodents, we found that local exposure to CB1R agonist within the first postnatal week, but not thereafter, led to a decline in the induction efficacy of long-term depression at GABAergic synapses (LTDGABA), a key step in the hard-wiring of vestibular circuits. Within this critical period, endocannabinoid-mediated LTDGABA at inhibitory neurons was selectively triggered by cholecystokinin, whereas that at excitatory neurons was by serotonin. Neonatal exposure to cannabinoids extended the phase of high GABAergic synaptic plasticity and overrode the synapse-specific, modulatory mechanism for plasticity. Such treatment delayed the postnatal emergence of vestibular-dependent reflexes and deranged adult navigational behavior. Deficits in higher functions are thus attributable to the maldevelopment of sensory processing circuits resulting from early cannabis exposure.
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Subject areas: Natural sciences, Biological sciences, Neuroscience
Highlights
- •Early cannabis exposure impairs graviceptive reflexes via presynaptic CB1R
- •CB1R activity in early postnatal stages shapes vestibular GABAergic plasticity
- •Disinhibitory and inhibitory sites use distinct triggers for eCB-mediated plasticity
- •ECB-mediated LTDGABA alters the development of navigation
Teaser
Natural sciences; Biological sciences; Neuroscience
Article notes
Untitled section
Received 2024 Mar 12; Revised 2025 Feb 10; Accepted 2025 Apr 28; Collection date 2025 Jun 20.
Introduction
Sequential emergence of increasingly complex behavior in development involves nature-defined innate developmental programs subject to nurture resultant from experience-dependent fine-tuning of neural circuits mediated by synaptic plasticity.1 The endocannabinoid system (eCB) plays crucial roles in modulating plastic processes that refine neural circuits based on experience.2 Of particular importance is the action of the eCB system on the plasticity of gamma-aminobutyric acid (GABA)ergic transmission, which in turn sets the level of neural plasticity in neonatal brain circuits. We asked how aberrations in neonatal GABAergic transmission could lead to circuit dysfunction, manifesting as behavioral deficits in patients with perinatal overexposure to cannabinoids.3 Vestibular circuits, which undergo significant refinement in the early postnatal period,4,5,6 are most susceptible to early cannabinoid exposure.
The role of GABAergic neurons in shaping afferent input processing at second-order vestibular neurons was established by pioneering studies in the frog.7,8 Previously, we showed that GABAergic transmission was required for the functional maturation of vestibular circuits,4,5 even with intact inputs from vestibular sensory input.6 In this study, we further demonstrate that type I cannabinoid receptor (CB1R) activity in the early postnatal stage sets the trajectory of synaptic plasticity at GABAergic synapses of vestibular nucleus (VN) circuits.
High synaptic plasticity in GABAergic transmission within the critical period and abrupt decrease of plasticity at the end of the critical period is a common mechanism for the fixation of synaptic characteristics in many sensory systems after a postnatal period of input-dependent refinement.9,10,11 While it is assumed that a period of heightened plasticity within the critical period allows input-guided fine-tuning of developing circuits,10 it is not known whether the temporal control of plasticity in GABAergic transmission is required for circuit maturation. In the absence of sensory input, such as during dark rearing, eCB was required for closure of the extended critical period.12 Given omnipresent vestibular input is present in neonate4,13,14 and neuronal expression of CB1R since birth,15 we asked how the activity of the eCB system early in the critical period determines the closure of the critical period.
Vestibular outputs are conveyed via distinct projection pathways segregated by function,13,14 with distinct output dynamics.16 This implied existence of distinct signal processing circuits within the VN. Evidence suggests that such differences in the processing of sensory inputs arise not only from differing intrinsic properties of neuronal subtypes,17 but also from differences in inhibitory inputs.7 On the most basic level, inhibitory synapses can be classified as inhibitory motifs when GABAergic neurons impinge on excitatory neurons, or disinhibitory motifs when both the pre- and postsynaptic neurons are inhibitory.18 By separating these two motifs during the recording of GABAergic postsynaptic currents in VGAT-Venus mice,19 we revealed type B cholecystokinin receptors (CCKBR) and serotonin-2A receptors (5-HT2AR) as unique physiological triggers of eCB-mediated plasticity at developing disinhibitory and inhibitory motifs, respectively.
Here, we show that early postnatal exposure to CB1R agonist delayed the maturation trajectory of GABAergic plasticity in VN circuits in rodents, with such animals suffering from long-lasting navigational deficit in adulthood. Although the role of motif-specific tuning of plasticity during the critical period remains to be elucidated, behavioral deficits resulting from overriding these triggers by the non-discriminatory stimulation of eCB receptors revealed its importance in circuit maturation. Results, therefore, highlight the importance of both temporal and motif-specific regulation of LTDGABA toward the formation of functional circuits in the VN.
Results
A critical period for endocannabinoid modulation of graviceptive behavior
Localized efflux of CB1R agonist WIN55 above the VN from P1 delayed the emergence of negative geotaxis to P13 (Figure 1A1, WIN55-treated vs. sham controls, a blocking peptide against calcineurin20,21,22 (CaN-BP)- or CB1R antagonist AM251-treated at P13: p < 0.001), contrasting with saline controls in which this behavior emerged by P9. The air righting reflex, which normally emerges at P17, was also delayed to P18 by such treatment (Figure 1A2, WIN55-treated vs. all other treatments at P17: p < 0.01). Contrarily, exposure to AM251 from P1 advanced the emergence of negative geotaxis to P8 (Figure 1A1, AM251-treated vs. sham controls, WIN55-, or BIC-treated at P8: p < 0.001). The combined treatment of WIN55 and AM251 had no significant effect on either behavioral test (Figure S2). This established that CB1R activity in the early postnatal stage bi-directionally affected the circuit maturation of the medial vestibular nucleus (MVN).
Exposure to WIN55-loaded Elvax from P12 onwards did not affect negative geotaxis (Figure S3B). This time point coincided with the switch in polarity of GABAergic transmission (EGABA) in VN from depolarizing to hyperpolarizing at P12 (Figure 1B), a hallmark of the maturation of GABAergic circuits.23 We further found that WIN55 exposure at P8, one day before the functional maturation of circuits for negative geotaxis, also caused no delay (Figure S3A). Results corroborate clinical evidence that chronic adult exposure to cannabinoids is largely inconsequential24 and highlighted a critical period closing at P8 during which the CB1R directed maturation of VN circuits.
Decrease in induction efficacy of long-term depression at GABAergic synapses drives circuit maturation
Decreased synaptic plasticity of VN circuits in normal rats at P8, as reflected magnitude of long-term change in synaptic strength after TBS (Figure 1C1), accompanied closure of the critical period for vestibular development at this stage.
Examination of individual responses revealed a significant decrease in percentage of MVN neurons displaying an LTD response after TBS, from 80% within the critical period (P5−8) to 20% after the closure of the critical period for VN circuits that support reflexive actions at P9–11 (Figure 1C3, p < 0.001 vs. P5–8). The magnitude of LTDGABA in cells that continued to show LTD response in P9–11 rats was also reduced compared to those in P5–8 rats (63.46 ± 9.03%, n = 6/26 cells, 10 rats, p = 0.002) (Figure 1C3). Addition of GABAA receptor antagonist bicuculline to the bath after the LTD measurements confirmed that the PSCs observed were indeed mediated by GABAA receptors (Figure S3D). These confirmed that the maturation of MVN circuits and closure of the critical period were also marked by a decrease in synaptic plasticity, as in critical periods of cortical circuits.25
We then tested whether decreased LTDGABA incidence could, in turn, promote the functional maturation of VN circuits. Exposure of VN neurons to CaN-BP at P1 decreased LTDGABA incidence in the neonatal VN to 15% (Figure 1C, p < 0.001 vs. untreated). Such treatment also advanced the emergence of negative geotaxis from P9 to P7 (Figure 1A1), and that of air righting behavior from P17 to P15 (Figure 1A2). Contrarily, blockade of GABAA receptors to simulate depressed GABAergic transmission resultant from sustained high LTDGABA incidence, using a bicuculline-loaded Elvax slice, delayed the emergence of negative geotaxis to P15 (Figure 1A1) and that of air righting reflex to P18 (Figure 1A2). These results suggested that the plasticity of GABAergic transmission is causal for VN circuit maturation.
Long-term depression at GABAergic synapses in the medial vestibular nucleus during the critical period is mediated by endocannabinoid signaling
To dissect the controlling mechanism for LTDGABA in the VN during the early postnatal period (P5–8), we first determined if pre- or post-synaptic mechanisms were dominant in this period. Significantly increased miniature PSCGABA frequency (0.46 ± 0.03 Hz to 0.64 ± 0.04, n = 19 cells, p < 0.01) and decreased paired pulse ratio (PPR, 1.49 ± 0.39 to 1.15 ± 0.29, n = 19 cells, p < 0.001) after TBS (Figure 1C2), and unchanging PSCGABA amplitude (before TBS: 38.64 ± 0.82 pA, after TBS: 35.55 ± 0.91 pA, n = 19 cells, p = 0.874) pointed to the pre-synaptic regulation of GABAergic plasticity.
We therefore tested if the presynaptic CB1R receptor12,18,26,27 was a major regulator for synaptic plasticity in the neonatal VN. Blocking synthesis of CB1R ligand 2-arachidonoylglycerol (2-AG) with Orlistat decreased the percentage of P5–8 MVN neurons capable of expressing LTDGABA from 80% (control) to 38% (Figures 1C3 and 1D, p = 0.008 vs. untreated). Conversely, the inhibition of 2-AG degradation with JZL184 in P9–11 MVN neurons increased the induction efficacy of LTDGABA from 29% (control) to 78% (Figures 1C3 and 1E, p < 0.001 vs. untreated). It is noteworthy that JZL184 caused a decrease in membrane resistance (Figure S4). These pharmacological manipulations proved that the majority of TBS-induced LTDGABA in the neonatal rat VN was elicited by activity of eCB system.
Early cannabinoid exposure prolonged high level of long-term depression induction at GABAergic synapses
If so, can CB1R activity set the developmental trajectory of decreasing LTDGABA incidence? Abolishment of further LTDGABA response to a second TBS by the bath addition of AM251 (Figure 2A; Figure S5) or Orlistat (Figure 2F, blue tracing) proved that the observed LTDGABA was indeed mediated by CB1R. In rats pretreated with WIN55 at P1, a high incidence (79%) of eCB-mediated LTDGABA (Figure 2B) was maintained to P9–11 (Figure 2E, 78%), contrasting with 23% in controls (Figure 2E, p < 0.001 vs. sham controls). Conversely, pre-treatment with AM251 at P1 decreased the occurrence of eCB-mediated LTDGABA at P5–8 from 79% (sham) to 23% (Figure 2B, p < 0.001 vs. sham), indicating the premature suppression of plasticity in MVN circuits. The amplitude of LTD in cells that responded as such to TBS, however, was unchanged by pre-treatment with WIN55 at P1 (46.84 ± 7.83%, n = 17/23 cells, 11 rats, p = 0.814) or AM251 at P1 (45.42 ± 7.09%, n = 8/41 cells, 12 rats, p = 0.934) as compared to control rats.
In P9–11 rats, the lower percentage of cells showing LTDGABA after TBS was not due to the developmental downregulation or inactivation of CB1R on these neurons. Using a double TBS protocol, P5–8 or P9–11 MVN neurons which did not display LTDGABA after an initial TBS could be induced to display LTDGABA by increasing synaptic 2-AG concentration with JZL181 (Figure 2C, purple tracing) or direct activation of CB1R with WIN55 (Figure 2D). Additionally, most recorded neurons were excitatory, and WIN55 decreased their excitability (Figure S6). This implied that the retrograde activation of CB1R by postsynaptic release of endocannabinoids was the predominant factor that governed the incidence of LTDGABA in the VN.
Unique triggers for 2-arachidonoylglycerol release in medial vestibular nucleus circuits
We then tested the efficacy of other Gq-coupled receptors, such as 5-HT2AR and CCKBR,28,29 that were less commonly associated with 2-AG release in the forebrain.30,31 Bath application of 5-HT2A receptor antagonist MDL11939 (Figure 3A1) significantly decreased the probability of LTDGABA incidence among P5–8 MVN neurons to 30% (p = 0.004), while CCKBR antagonist CI988 (Figure 3A2) produced a slight decrease to 50% (p = 0.096) (Figure 3A4). While the same canonical PLC-, PKA-, and Ca2+-dependent intracellular pathways as those in forebrain neurons32,33 were utilized by MVN neurons to effect eCB mediated LTDGABA (Figures S7A–S7C), MVN neurons utilized distinct transmembrane signaling receptors to trigger 2-AG release. In the forebrain, 2-AG release triggered by metabotropic glutamate receptor (mGluR) is well documented.34,35,36 Bath application of mGluR5 antagonist MPEP or mGluR1 antagonist LY367385 to MVN slices of P5–8 rats (Figure 3A3), however, did not change the induction efficacy of eCB-mediated LTDGABA (Figure 3A4, MPEP, p = 1; LY367385, p = 0.347 vs. control).
Whereas in P9–11 rats, the application of 5-HT2R agonist α-m-5-HT (Figure 3B1) significantly increased the proportion of cells responding to TBS with LTDGABA to 78% (p < 0.001 vs. control), while CCK (Figure 3B2) increased the proportion to 57% (p = 0.011 vs. control) (summarized in Figure 3B4). Such an increase in the proportion of cells showing LTDGABA response was confirmed to be eCB-mediated by the addition of orlistat to the bath which blocked further induction of LTDGABA by a second TBS in P9–11 rat VN neurons despite the presence of CCK (Figure 3B2) or α-m-5-HT (Figure 3B1). The apparent redundancy of 5-HT2AR and CCKBR on triggering eCB-mediated LTDGABA in the MVN suggested the possibility of yet unknown mechanisms for differential control of plasticity in GABAergic transmissions. Addition of mGluR1 agonist DHPG (Figure 3B3) also did not affect the induction of eCB-mediated LTDGABA in P9–11 cells (Figure 3B4, p = 0.484 vs. control). These results thus demonstrate that the activation of mGluR1/5 did not trigger postsynaptic release of 2-AG as a retrograde messenger necessary for the induction of eCB-mediated LTDGABA in MVN, distinct from glutamate-dependent triggering eCB-mediated LTDGABA in the hippocampus.37
Cell-type specific control of long-term depression at GABAergic synapses by the differential triggering of endocannabinoid release
GABAergic neurons impinging upon excitatory or inhibitory neurons form inhibitory and disinhibitory motifs, respectively.18 Being the building blocks of functional circuits,18 we reasoned that the tuning of plasticity at such motifs would require distinct regulation. Use of vesicular GABA transporter (VGAT)-Venus transgenic mice19 allowed the distinction of plasticity at disinhibitory (Figure 4A1) and inhibitory motifs (Figure 4A2). The overall profile of TBS-induced neuronal plasticity in VGAT-Venus mice was not different from rats (P5–8 VGAT mice vs. P5–8 rat, p = 0.682; P9–17 VGAT-Venus mice vs. P9–11 rat, p = 0.694) nor from wild type mice (P5–8, p = 0.402; P9–17, p = 1).
Moreover, VGAT and non-VGAT neurons had similar incidence of LTDGABA (P5–8, p = 0.538; P9–17, p = 0.873, Figures 4A1,2, and 4G1,2), which were reduced with age (VGAT neurons, p < 0.001; non-VGAT neurons p = 0.033). The amplitude of LTDGABA after TBS was also similar between VGAT-Venus and rats (P5–8, p = 0.779; P9–17, p = 0.580). Addition of Orlistat could abolish further induction of LTDGABA in both VGAT (Figure 4C1) and non-VGAT cells (Figure 4C2) in P5–8 MVN VGAT-mice. These confirm the validity of VGAT-Venus mice as a model for studying LTDGABA in the MVN during early postnatal development.
We revealed the differential modulation of LTDGABA incidence at VGAT versus non-VGAT neurons by the bath addition of 5-HT2AR or CCKBR agonists/antagonists. In P5–8 MVN slices, the bath addition of 5-HT2AR antagonist MDL11939 did not change the induction efficacy of LTDGABA in VGAT neurons compared to untreated controls (Figures 4D1 and 4F1, p = 0.560), but such treatment reduced LTDGABA incidence to 17% in non-VGAT neurons (Figure 4D2, p = 0.008). While the effect of CI988 was not significant in random patch-clamp recordings of MVN neurons (cf. Figure 4A2), cell type-specific recording in P5–8 VGAT-Venus mice revealed that CCKBR antagonist CI988 significantly decreased the probability of LTDGABA induction from 94% to 50% in VGAT neurons (Figures 4E1 and 4G1, p = 0.009), but not in non-VGAT neurons (Figures 4E2 and 4G1, p = 0.294). Additionally, the combined bath application of MDL11939 and CI988 did not suppress LTDGABA induction more effectively than either antagonist alone (Figure S8).
Distinct triggering of eCB-mediated plasticity at inhibitory and disinhibitory motifs persisted after closure of the critical period. In P9–17 MVN, 5-HT remained as a trigger for LTDGABA in non-VGAT neurons. Increasing the proportion of LTDGABA responses from 20% to 87% (p = 0.001, Figure 4F2) but not in VGAT neurons (from 20% to 19%, p = 0.227, Figure 4F2). On the other hand, the bath addition of CCK continued to increase the proportion of cells responding to TBS with LTDGABA in VGAT neurons (from 31% to 73%, p = 0.004, Figures 4F1 and 4G2) but not non-VGAT neurons (from 20% to 31%, p = 0.857, Figures 4F2 and 4G2). In all, results revealed that the triggering of LTDGABA in non-VGAT neurons was 5-HT2AR-dependent while that in VGAT neurons was CCKBR-dependent (Figure 4H), and this phenomenon is calcium-independent (Figure S9).
Long-term plasticity in cannabinoid receptor 1 knock-out mice
The role of eCB system on GABAergic synaptic transmission and plasticity was further confirmed using CB1R knock-out (Cnr1−/−) mice. The amplitude of miniature IPSCGABA was similar between control and knockout mice both at P5–8 (Figure 5A1,2, p = 0.14) and P9–17 (Figure 5B1,2, p = 0.83). Decay time of mPSCGABA was shorter in P5–8 Cnr1−/− mice compared to controls (Figure 5A2, p = 0.037). The frequency of mPSCGABA in Cnr1−/− mice was significantly lower than age-matched controls (Figures 5A2 and 5B2, P5–8: p = 0.014, P9–17: p = 0.04) with longer inter-event interval (Figures 5A1 and 5B1, p = 0.03) throughout the period investigated (P5–17). This decreased probability of GABA release suggested less depression of GABAergic transmission in Cnr1−/− mice. Moreover, significantly increased PPR in Cnr1−/− mice throughout this period compared to controls (Figures 5A2 and 5B2, P5–8: p < 0.0001, P9–17: p < 0.0001) supported the notion of a presynaptic site of action for CB1R.
Whole-cell patch-clamp recording from MVN neurons in Cnr1−/− mice further confirmed the role of CB1R on LTDGABA induction. Incidence of LTDGABA was significantly lower in P5–8 Cnr1−/− mice (44% in Cnr1−/−, Figure 4G1) compared to VGAT-Venus mice (88%, p = 0.001). After the maturation of VN circuits at P9–17, incidence of LTDGABA was no longer different between Cnr1−/− (7%, Figure 4F2) and VGAT-Venus mice (27%, p = 0.07) mice after the maturation of VN circuits. These results highlight the importance of CB1R in controlling LTDGABA response among developing VN circuits.
Long-lasting effects of neonatal perturbation of type I cannabinoid receptor in the vestibular nucleus on the maturation of spatial navigation
Direct dosing of CB1R agonist and antagonist bypassed motif-specific regulatory mechanisms for 2-AG release, with corresponding alteration to the duration of the neonatal period of high induction efficacy of LTDGABA, and were accompanied by shifts in the maturation of MVN circuits for reflexes. Given that VN outputs also inform spatial cognition, we reasoned that the perturbed maturation of MVN circuits further impacts multimodal functions such as spatial cognition.14 The dead reckoning test was used to assess the spatial cognition of adult rats38 (Figure S10). In the dark probe test, where visual signals are absent, vestibular signals become the dominant sensory input for navigational behavior.38 Sham control rats acquired the task significantly faster than adult rats pretreated with WIN55 at P1 (Figure 6E, p < 0.01). Analysis of the pattern of homeward paths of all WIN55-pretreated rats (Figure 6A, third column from the left), revealed deficits in homeward navigation (i.e., increase in heading angle, time spent in the food quadrant, and error in locating the homebase) in the dark (p < 0.01 for all parameters). Normal performance of these rats in the light probe test supported a vestibular origin for the deficit (Figures 6B−6D). Early suppression of LTDGABA induction efficacy with CaN-BP (Figure 1C) or AM251 pretreatment at P1 (Figure 2B) did not cause navigational impairment in either the light or dark probe test (Figures 6A–6D). However, when spatial memory played a more significant role, such as when presented with conflicting visual and vestibular cues in the new location test, both WIN55- and AM251-pretreated rats had difficulty finding their way back to the new home (Figures 6A−6D).
Absence of navigational deficits both in the light and dark probe tests despite perturbation with WIN55 or AM251 at P8 or P12 implied the normal operation of adult circuits for processing visual and vestibular inputs (Figures S11 and S12). This demonstrated that the critical period for vestibular-dependent navigation was also closed by P8.
Discussion
Retardation of neurodevelopment by early exposure to cannabinoids has been documented3 but the mechanism of this has remained unclear. By following the maturation of the vestibular system in the early postnatal stage, this study provides evidence that control of LTDGABA by CB1R is crucial not only for the timely maturation of local reflexive circuits in the MVN but also permanent establishment of vestibular-dependent higher functions such as navigation. We revealed specific triggers for LTDGABA at inhibitory and disinhibitory motifs within the MVN. Bypassing these triggers with the non-discriminatory activation of presynaptic CB1R in the early postnatal period sustained high occurrence of plasticity in GABAergic transmission beyond the normal duration of the critical period. This led to delayed postnatal emergence of vestibular-dependent reflexes and deficits in adult spatial cognitive performance. Taken together, the results provide a mechanistic link between early cannabinoid exposure and neurodevelopmental deficits.
Bidirectional impact of neonatal endocannabinoid system activity on maturation profile of graviceptive reflexes
GABAergic neurons play a key role in the processing of afferent inputs in VN circuits.5,6,7,8 While temporal control of key electrophysiological events, such as excitatory-inhibitory switch of GABAergic transmission in the forebrain39 are known to affect maturation, the contribution of temporal control of the plasticity in GABAergic transmission remains unclear. We show that long-term plasticity in GABAergic transmission within the MVN circuits is attenuated by the end of the critical period at P8, similar to other sensory systems.11,40,41 This, in theory, allows higher levels of depolarizing GABAergic transmission to provide the excitatory drive for the consolidation of neuronal circuits,23,42,43 as well as prevent further activity-dependent pruning of synapses.
CB1R activity is known to be subject to homeostatic regulation, including decreased receptor expression44 and phosphorylation of the cytoplasmic tail of the receptor upon repeated dosing of WIN55 over a few days.45,46 However, such desensitization is dependent on both age and brain location, with cerebellar neurons of adolescent rats showing non-significant levels of desensitization after prolonged cannabinoid dosing.47 While desensitization was not investigated in this study, the desensitization of CB1R in MVN after WIN55 dosing, if present, would further highlight the sensitivity of early postnatal M VN circuit to transient perturbations in CB1R activity.
In addition, we have observed that P9-17 Cnr1−/− mice expressed LTP in MVN neurons (Figure 4B; Figure S13). This suggests that normal CB1R activity is critical for the timely regulation of LTDGABA and, by extension, for the proper development of vestibular circuits. Furthermore, the absence of CB1R disrupts the balance between LTP and LTD, which normally helps to refine synaptic connections during development.1 LTP in these mice might result from an unregulated, excessive excitatory drive or from compensatory mechanisms due to the loss of CB1R-mediated inhibition. This unregulated plasticity could disrupt normal circuit development, potentially mirroring or exaggerating the effects seen with early cannabinoid exposure.
Interestingly, decay time of mPSCGABA was shorter in P5–8 Cnr1−/− mice compared to controls (Figure 5A). Presynaptic CB1 receptors influence postsynaptic response as evidence show that endocannabinoids selectively inhibit a subclass of synapses distinguished by their fast kinetics and large unitary conductance.48 Cnr1−/− mice which lack CB1 receptors might lead to compensatory changes in the expression or subtype composition of GABA receptors. There might be an increase in the expression of GABAA receptor subtypes that have faster desensitization kinetics, which could contribute to the shorter decay time.
5-HT2AR and CCKBR are involved in the retrograde regulation of long-term depression induction in vestibular nucleus
Having established a role for eCB in modulating the development of early postnatal MVN, we further asked how eCB release itself is regulated to suit circuit-specific needs. MVN neurons utilize 5-HT2A and CCKB receptors to trigger activity-dependent release of 2-AG instead of the more common mGluR. These dual triggers allowed LTDGABA at disinhibitory motifs to be modulated specifically by CCKBR, whereas that at inhibitory motifs by 5-HT2AR. Thus far, inhibitory dynamics have only been described for second order VN neurons in the mature animal,7,8,49 with little known about the function and maturation processes of disinhibitory motifs within the VN. Due to non-specific depolarization resultant from bipolar electrode stimulus and the placement of electrodes at the medial margin of the MVN,50,51 GABA-on-GABA inputs to the VN such as those from commissural inhibition,49 ipsilateral GABAergic neurons outside the VN similar to those for glycinergic inputs,52 or local VN interneurons could not be differentiated. Nonetheless, results revealed a layer of complexity for plasticity in the processing of vestibular information beyond previously reported synaptic plasticity in excitatory inputs to GABAergic versus non-GABAergic VN neurons.53
In a normal developing system, CCK and 5-HT work in a coordinated manner to regulate LTDGABA at inhibitory and excitatory synapses, respectively. This coordination ensures a balanced development of the vestibular circuits and proper sensory processing. With early cannabinoid exposure, this coordination is disrupted. The failure to synchronize CCK and 5-HT activity properly results in improper circuit formation and associated behavioral deficits.
Limitations of the study
The origin of GABA-on-GABA inhibitory inputs was not revealed under the current stimulation paradigm. Nonetheless, results revealed a layer of complexity for plasticity in the processing of vestibular information beyond previously reported synaptic plasticity in excitatory inputs to GABAergic versus non-GABAergic VN neurons.
Behavior-dependent recruitment of distinct subpopulations of MVN neurons to various vestibulo-thalamocortical pathways remains unclear. More importantly, molecular or electrophysiological evidence for distinct critical periods in such subpopulations awaits further investigation. Our observations provided behavioral evidence that VN neurons that support higher centers involved in complex multi-modal tasks have a later closure date in critical periods as compared to those supporting reflexive actions. Given that real world tasks are more complex than standardized behavioral tests, we expect the sensitivity period to exogenous cannabinoid exposure to extend into the juvenile stage in humans.
Resource availability
Lead contact
Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Ying-Shing Chan (yschan@hku.hk).
Materials availability
This study did not generate new materials.
Data and code availability
- •Raw data may be obtained from the corresponding author upon reasonable request.
- •No code was generated from this work.
- •All reagents, chemicals, and behavioral apparatus were either obtained commercially or could be assembled according to the methods using freely available materials.
Acknowledgments
We thank Professor Yuchio Yanagawa for VGAT-Venus mice; Simon S.M. Chan for constructing the setups for negative geotaxis, air righting reflex, dead reckoning test, and in vitro electrophysiological recording; Alice Y.Y. Lui, Tony Xiaotong Liang and Florence Yu Sum Keung for assistance in the preparation of Elvax and behavioral tests; Kimmy F.L. Tsang for assistance with histology and radioisotope work. This work was funded by Beijing Natural Science Foundation (Grant No. Z200024, Z240011 and L222097 to W.S.), Beijing Hospital Authority Clinical Medicine Development Special Fund (ZLRK202333 to W.S.), Hong Kong Research Grants Council (GRF 761812 to D.K.Y.S.and Y.S.C.), HMRF Grant (06172866 to K.L.K.W. and Y.S.C.), State Key Laboratory of Brain and Cognitive Sciences, HKU, and the Chi Lin Kok Ng BHL Foundation.
Declaration of interests
The authors declare that they have no competing interests.
STAR★Methods
Key resources table
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Chemicals, peptides, and recombinant proteins | ||
| WIN55 ((3R)-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl)-1-naphthalenyl-methanone, monomethanesulfonate) | Tocris Bioscience 1038/10 | |
| CNQX | Tocris Bioscience 0190/10 | |
| D-AP5 | Tocris Bioscience 0106/1 | |
| FK506 | Tocris Bioscience 3631 | |
| BIC (bicuculline methiodide) | Tocris Bioscience 0131/10 | |
| TTX | Sigma-Aldrich 554412 | |
| AM251 (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) | Tocris Bioscience 1117/1 | |
| SR141716A (N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) | Tocris Bioscience 0923/10 | |
| BAPTA AM | Tocris Bioscience 2787/25 | |
| 7,8-dihydroxyflavone (7,8-dihydroxy-2-phenyl-4H-1-benzopyran-4-one) | Tocris Bioscience 3826/10 | |
| MPEP (2-methyl-6-(phenylethynyl) pyridine hydrochloride) | Tocris Bioscience 1212/10 | |
| K252a ((9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2',1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester) | Tocris Bioscience 1683/200U | |
| LY367385 ((S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid), DHPG (RS)-3,5-dihydroxyphenylglycine | Tocris Bioscience 1237/10 | |
| JZL184 (4-[Bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) | Tocris Bioscience 3836/10 | |
| Orlistat (N-formyl-L-leucine (1S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]dodecyl ester) | Tocris Bioscience 3540/10 | |
| CCK (cholecystokinin) | Tocris Bioscience 1166/1 | |
| CI988 | Tocris Bioscience 2607/10 | |
| U73122 (1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) | Tocris Bioscience 1268/10 | |
| KT5720 ((9R,10S,12S)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2',1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, hexyl ester) | Tocris Bioscience 1288/100U | |
| MDL11939 (α-Phenyl-1-(2-phenylethyl)-4-piperidinemethanol) | Tocris Bioscience 0870/10 | |
| α-m-5HT (α-Methyl-5-hydroxytryptamine maleate) | Tocris Bioscience | |
Experimental model and study participant details
Animals
Sprague-Dawley rats (Charles River Lab), C57Bl6/J mice (Jackson Laboratory), vesicular GABA transporter (VGAT)-Venus transgenic mice19 (a gift from Professor Y Yanagawa, Gunma University Graduate School of Medicine), and CB1R knockout (Cnr1−/−) mice (Shanghai Model Organisms Center Incorporated) were used. For experiments conducted on adults, only male rats were used. Early postnatal animals were randomly picked for electrophysiological and behavioral experiments as the sex of rodents prior to weaning is not explicit. Procedures were approved either by The University of Hong Kong Committee on the Use of Live Animals in Teaching and Research or by Beihang University Ethics Review Board.
Method details
Surgery implantation of Elvax slice
200 μL of 10 mM CB1 receptor agonist WIN55 and/or 10 mM CB1 receptor antagonist AM251 solution in dimethyl sulfoxide (DMSO, 4%, Sigma) were mixed and snap frozen with a 10% (w/v) Elvax solution in dichloromethane as described previously. Solidified slices were kept at −20 C to allow evaporation of dichloromethane. Slices cut to final dimensions (1 mm × 1 mm, 200 μm thickness) prior to implantation to the 4th ventricle via the foramen magnum.
Dead reckoning behavioral test
Dead reckoning test for spatial cognition was conduction on P60 rats implanted with drug-loaded Elvax slices at P1, P8 or P12, and sham operated rats. Rats were fasted for 12 h prior to test sessions. Only one food pellet (1 g, Supreme Mini-Treats, Bio-Serv) was provided during each test trial. Rats foraged for the food pellet placed randomly around the middle of the circular arena before returning to their home cage in 1 of the 8 possible locations around the arena. Stationary visual cues were provided during the training sessions and light probe tests. In the dark probe test, the lights were switched off and the arena was surrounded completely by a ceiling-to-floor black curtain. The new home location test was conducted in light with the home base moved to the hole diametrically opposite to its original home base. Eight trials were done in each of the light or dark probe tests and 4 in the new location test spread over 3 consecutive days (Figure S10). Heading angle, time in the quadrant with food, errors the rats made in return path, and the training time needed before rats learnt the task were measured from recorded video footage.
Patch clamp recording
Borosilicate glass pipettes (4–6 MΩ) filled with internal solution for voltage clamp containing (in mM): 140 KCl, 2 MgCl2, 2 Na2ATP, 1 ethylene glycol-bis (b-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (EGTA), and 10 N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES) (adjusted to pH 7.3, 285–295 mOsm) were used. KCl-based internal solution was used to record evoked GABAergic postsynaptic currents (ePSCGABA). For current-clamp recordings, electrodes were filled with an internal solution containing the following (in mM): 134 K-gluconate, 6 KCl, 10 HEPES, 4 NaCl, 7 K2-phosphocreatine, 0.3 NaGTP, and 4 Mg-ATP (pH 7.3 adjusted with KOH). No series resistance compensation was applied but the cell was discarded if the access resistance changed significantly (>25%) during the course of recording. Cell recording was discarded if the leaking current was >100 pA.
Quantification and statistical analysis
The statistical analyses were performed using GraphPad Prism 9 software. All statistical details of the experiments can be found in the figures and figure legends. All data are presented as mean ± SEM. One-way ANOVA followed by Bonferroni’s correction for multiple comparison was used to compare the average time for accomplishing positive responses in negative geotaxis and air righting tests, as well as performance indexes in the dead reckoning test. Differences with p < 0.05 were considered statistically significant.
Untitled section
Published: April 30, 2025
Footnotes
Footnote Group
Contributor Information
Daisy Kwok-Yan Shum, Email: shumdkhk@hku.hk.
Ying-Shing Chan, Email: yschan@hku.hk.
Supplemental information
References
Untitled section
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Associated Data
Supplementary Materials
Data Availability Statement
- •Raw data may be obtained from the corresponding author upon reasonable request.
- •No code was generated from this work.
- •All reagents, chemicals, and behavioral apparatus were either obtained commercially or could be assembled according to the methods using freely available materials.