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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Neuron. Author manuscript; available in PMC 2011 April 29.
Published in final edited form as:
PMCID: PMC2897012
NIHMSID: NIHMS199496

The maturation of GABAergic transmission in visual cortex requires endocannabinoid-mediated LTD of inhibitory inputs during a critical period

SUMMARY

Endocannabinoids are widely regarded as negative modulators of presynaptic release. Here we present evidence that in visual cortex endocannabinoids are crucial for the maturation of GABAergic release. We found that between eye opening and puberty, release changes from an immature state with high release probability, short-term depression (STD) and high release variability during irregular patterned activity, to a mature state with reduced release probability, STD and variability. This transition requires visual experience and stimulation of CB1 cannabinoid receptors as it is mimicked by administration of CB1 agonists, blocked by antagonists and is absent in CB1R KO mice. In immature slices, activation of CB1 receptors induces long-term depression of inhibitory responses (iLTD), and a reduction in STD and response variability. Based on these findings, we propose that visually induced endocannabinoid-dependent iLTD mediates the developmental decrease in release probability, STD and response variability, which are characteristic of maturation of cortical GABAergic inhibition.

INTRODUCTION

Inhibitory GABAergic interneurons comprise 20 % of total cortical neurons (Peters and Kara, 1985), yet they significantly constrain neural activity patterns and propagation, and play a central role in cortical processes including synchrony (Bartos et al., 2007), attention (Mitchell et al., 2007) and plasticity (Jiang et al., 2005; Spolidoro et al., 2009). In sensory cortices, GABAergic circuits are not fully functional at birth, with maturation occurring slowly over development until completion at puberty. Indeed, this protracted maturation is believed to define an early postnatal critical period during which activity-dependent plasticity can be more effectively recruited by sensory experience (Hensch et al., 1998; Huang et al., 1999; Kirkwood and Bear, 1994). Moreover, experience-dependent plasticity might involve changes in GABAer circuitry (Maffei, et al 2006). Hence elucidating the mechanisms that underlie the development of cortical GABAergic circuits is fundamental for understanding cortical processing and synaptic plasticity.

At the synaptic level, the efficacy of fast GABAergic synaptic transmission is dictated by the number of presynaptic release sites, the presynaptic release probability and the postsynaptic response to released GABA. Prominent postsynaptic alterations include a switch in the polarity of the GABAergic response from depolarizing to hyperpolarizing, and a change in the subunit composition of the GABAA receptors (Heinen et al., 2004). However, both of these alterations in rodents are completed in early postnatal development, (Heinen et al., 2004; Luhmann and Prince, 1991) and are therefore unlikely to affect the regulation of GABAergic transmission after eye opening. Indeed, much of the developmental gain in GABAergic strength is attributed to a remarkable 2–3 fold increase in the number of GABAergic synapses from eye opening (circa 15 days in rodents) to puberty (circa 5 weeks). In visual cortex, this has been documented as an increase in the total number of perisomatic GABAergic contacts onto a given pyramidal cell, as well as the number of contacts made by an interneuron (Chattopadhyaya et al., 2004; Huang et al., 1999; Morales et al., 2002). Similar developmental increases in the number of GABAergic synapses have been documented in other sensory cortices and species (Gao et al., 1999; Micheva and Beaulieu, 1995). In visual cortex GABAergic synaptogenesis is triggered by sensory experience (Chattopadhyaya et al., 2004; Huang et al., 1999; Kreczko et al., 2009; Morales et al., 2002) and regulated by several permissive and suppressive mechanisms (Huang and Scheiffele, 2008).

The functional maturation of these newly formed GABAergic synapses is less understood. Curiously, evidence suggests that in visual cortex the probability of release at inhibitory synapses is reduced with postnatal development (Jiang et al., 2005; Morales et al., 2002; Tang et al., 2007) which superficially is at odds with the notion of a developmental increase in GABAergic inhibition. This developmental reduction in the probability of release appears to require normal visual experience, in that it is attenuated by dark rearing (Jiang et al., 2005; Tang et al., 2007). However the exact plasticity mechanisms recruited by visual experience to modify GABAergic release remain unknown.

Synaptic plasticity of the strength of GABAergic transmission has been documented in visual cortex (Komatsu, 1994; Maffei et al., 2006), although the previously described mechanisms do not appear to affect presynaptic release. Research in recent years underscored an essential role for endocannabinoid signaling in the plasticity and regulation of presynaptic release. Endocannabinoids are retrograde messengers that are produced and released postsynaptically in an activity-dependent manner and bind to presynaptic receptors that negatively regulates presynaptic release probability (Chevaleyre et al., 2006; Kano et al., 2009; Lovinger, 2008).

Endocannabinoid-dependent forms of long term depression of inhibitory transmission (termed iLTD) have been described in the amygdala (Marsicano et al., 2002) and in the CA1 region of the hippocampus (Chevaleyre and Castillo, 2003), and similar mechanism may underlie the decrease in release probability at inhibitory synapses over development in the visual cortex. To test this hypothesis we examined iLTD in layer II/III pyramidal cells of the visual cortex. We demonstrate that iLTD is essential for the maturation of presynaptic properties of GABAergic transmission and suggest that induction of iLTD may serve to enhance, rather than decrease, GABAergic function during periods of high neural activity.

RESULTS

Induction of a presynaptic endocannabinoid-dependent form of iLTD in the juvenile visual cortex

Pharmacologically isolated inhibitory postsynaptic currents (IPSCs) in layer II/III pyramidal cells were evoked by stimulation of layer IV. Theta burst stimulation (TBS, see Methods), previously shown to induce iLTD in the CA1 region of the hippocampus (Chevaleyre and Castillo, 2003), was used to induce iLTD (Figure 1). TBS induced a significant, sustained depression in the IPSC amplitude in cells from 3 week-old rats (77.3±1.3% of baseline at 30 min after TBS, n=53 from 45 rats, paired t-test: p<0.001, Fig 1C) that is robust (66% of the cases showed more that 20% depression, Fig 1D). To examine the locus of expression of iLTD we evaluated changes in paired pulse depression (PPD, inter-stimulus interval= 100 msec, see Methods), which provides a coarse estimate of the probability of release. After iLTD, PPD decreased from 0.23±0.025 to 0.16±0.031 (n=23, p<0.001, Fig 1E), suggesting that a reduction in presynaptic release probability underlies iLTD.

Figure 1
Theta burst stimulation of layer IV induces LTD of pyramidal cell IPSCs (iLTD) in layer II/III

In hippocampal CA1 and other systems (Chevaleyre et al., 2006) iLTD is initiated by the binding of endocannabinoids to type 1 cannabinoid receptor (CB1R). The endocannabinoids are produced and released postsynaptically in a process triggered by the activation of the mGluR5 metabotropic glutamate receptor, which is coupled to phospholipase C (PLC) (Chevaleyre and Castillo, 2003). To test whether a similar pathway mediates visual cortical iLTD, we studied the effects of CB1R ligands. Bath application of the CB1R antagonist AM251 prevented the induction of iLTD (10 μM AM251: 102.3±3.4%, 30 min after TBS n=6 rats, 10 cells; Control DMSO: 76.8±2.8%, n=6 rats, 7 cells, t-test: p<0.001, Fig. 1F). The effects of bath application of the CB1R agonist WIN55,212-2 were tested with a two pathway experiment (WIN: 10 μM, 10 min., 9 rats, 12 slices, Fig 1G). Inputs from one pathway were stimulated (paired pulses, 100 msec inter-stimulus interval at 0.03 Hz) during WIN application, and showed a sustained depression of the IPSCs that persisted when the agonist was “chased” with 10 μM AM251. Inputs from the second pathway were not-stimulated, and were not affected by WIN application (stimulated: 65.5±3.9%, 30 min after WIN: non-stimulated: 95.1±7.0%, p=0.004.t-test: p=0.004). The depression induced by 10μM WIN is blocked by AM251 (IPSC after 20 min in WIN: 98.0±6.1%, p=0.250) and absent in a CB1KO mice (Zimmer et al, 1999) line (105.1±3.7, n=3,7. p=0.16. Data not shown). Lower concentration of WIN (1μM) did not affect the evoked IPSCs (101.4±2.5, n=2,12, p=0.701). iLTD was also blocked by bath application of the mGluR5 antagonist MPEP (10 μM) (Control: 76.6±4.2%, n=6,6; MPEP 100.6±3.9%, n=6,10; p=0.001) and by the PLC blocker U73122 (10μM) (DMSO control 80.2±1.8%, n=5,6; U73122=98.3±2.1%, n=5,8; p<0.001. Fig. S1). Together this suggests that in the juvenile visual cortex, like in the CA1 region of the hippocampus, iLTD induction requires stimulation of CB1 receptors in conjunction with presynaptic activity (Heifets et al., 2008).

A critical period for iLTD induction

Critical aspects of GABAergic function, such as the number of perisomatic synaptic contacts onto pyramidal cells, continue to mature until puberty under the influence of normal visual experience (Chattopadhyaya et al., 2004; Kreczko et al., 2009; Morales et al., 2002). To examine the role of visual experience in the developmental regulation of iLTD we quantified iLTD in slices of visual cortex prepared from rats aged from 2 (p14–p20) to 10 weeks (p70-p76), reared either in normal light/dark cycles or reared in the dark from birth. In normal reared rats the iLTD magnitude was similar at 2 and 3 weeks of age (2 weeks: 74.6±2.3, n=7,13; 3 weeks: 75.9±1.7%, n=6,10), but was rapidly lost by the 5th week (99.1±1.3%, n=6,10; ANOVA: p<0.001. Fig 2B). In contrast, iLTD was still significant at 5 weeks in dark-reared rats (t-test: p<0.001 versus 5 week-old normal-reared rats. Fig. 2A), but a developmental loss occurred nonetheless, albeit later, between the 8th (77.2±5.5%, n=5,7) and the 10th (96.3±4.1%,n=6,9) week of age (ANOVA: p=0.002. Fig. 2B). These results indicate that in visual cortex iLTD can be induced only during a postnatal critical period with a duration regulated by sensory experience.

Figure 2
Induction of iLTD during a critical period regulated by visual experience

Visual experience is required for initiating the proliferation of GABAergic synaptic contacts, however, once this process is triggered, it is not stopped or reversed by visual deprivation (Chattopadhyaya et al., 2004; Morales et al., 2002)Therefore, we examined if visual deprivation could reverse the closing of the critical period for iLTD (Fig 2C, D). To avoid the potential confound of incomplete synaptogenesis, experiments were performed in rats older than 5 weeks, and dark rearing was initiated only after at least 3 weeks of age. In rats dark reared for 2 weeks starting at 3 weeks of age, the magnitude of iLTD was substantial (3N2D: 77.6±1.5%, n=6,10), comparable to the robust iLTD observed in normally reared 3 week-old rats (3N: 75.9±1.7%, n=6,10), and significantly larger than iLTD in normally reared 5 week old rats (5N: 99.1±1.3, n=6,10; F[2,27]=76, p<0.001). Similarly, when 4 week-old rats were placed in the dark for 1 week, the magnitude of iLTD measured was comparable to the declining iLTD observed in normally-reared 4 week-old rats (4N1D: 85.2±2.6%, n=5,11; 4N: 87.9±4.1%, n=8,14; p=0.56). If one week of dark rearing was started at 5 weeks of age, the magnitude of iLTD was unchanged, and was comparable to what is seen in 5 week-old rats (5N1D: 96.9±2.6%, n=4,6; p=0.510). These results suggest that visual deprivation can halt the developmental loss of iLTD at any moment during the critical period (Fig 2C,D). Finally, we examined the effects of re-exposure to light in dark-reared animals. We found that in rats dark reared for 2 weeks starting at 3 weeks of age, two days of light exposure was sufficient to “eliminate” iLTD (3N2D+L: 100.3±1.9%, n=8,14 compared with 3N2D: 77.6±1.5%, n=6,10; p<0.001. Columns 4 and 1 in Fig 2C, and downward arrow in Fig 2D). Subsequent reintroduction into dark for 2–6 days did not reactivate iLTD (3N2D+L+1D: 98.7±15,n=4,9, p=0.1. Column 5 in Fig 2C). In sum, the results suggest that visual experience drives the developmental loss of iLTD, which can be accelerated or retarded, but not reversed, by manipulating sensory experience during this period (Fig 2D).

To examine whether the presynaptic release probability and iLTD are co-regulated by age and visual experience, again we probed PPD. We confirmed previous reports (Jiang et al., 2005; Tang et al., 2007) that the magnitude of PPD decreased with age (F[3,36]=4.073; p=0.013) and found that this time course paralleled the developmental loss of iLTD (drawn as a grey line in Fig. 2E). Similar to the loss of iLTD, the developmental reduction in PPD was dependent on visual experience. PPD was larger in 5 week-old rats reared in the dark from birth (filled square in Fig. 2E) than in age-matched reared normal controls (DR: 26.1±2.7%; NR: 11.5±5.6%; p=0.031). Interestingly, despite initial differences in PPD across ages and rearing conditions, following the induction of iLTD with TBS, PPD was reduced to a similar value irrespective of age (thick line in Fig. 2E. F[4,45]=0.478; p=0.75).

The parallel developmental time courses suggest a casual link between the loss of iLTD and PPD. A simple scenario would be that neural activity evoked by vision causes iLTD in vivo, which reduces PPD. On the other hand, if iLTD occurs only in immature naïve synapses, the magnitude of iLTD would reflect the proportion of newly formed GABA synapses, which is high at 3 weeks of age but low at 5 weeks of age. Dark rearing, by preventing iLTD induction in vivo, would maintain the potential for inhibitory synapses to express iLTD.

Loss of sensitivity to endocannabinoids mediates developmental loss of iLTD

To explore the mechanisms involved in the loss of iLTD we set out to investigate which aspects of the CB1R signaling are altered by age. However, first we tested the possibility that the low release probability at 5 weeks (Fig 2E) may obscure the detection of presynaptic iLTD. We asked if enhancing the release probability by elevating extracellular [Ca2+] from 2 to 3.6 mM could rescue iLTD in slices from mature animals. Consistent with an increased release, raising the [Ca2+] increased both the IPSC amplitude (3 weeks: 152.2±12.7%, n=5,8; 5 weeks: 159.7±11.4%, n=6,8) and paired-pulse depression (3 weeks: 15.4±3.6%; 5 weeks: 22.9±3.4%) in cells from both 3 and 5 week-old rats (Fig. S2). However, iLTD was still insignificant in cells from 5 week-old rats (95.2±1.5%), and significant in cells from 3 week-old rats (77.6±2.8%, paired t-test versus baseline: p=0.003), confirming that the loss of iLTD with development is not due to the inability to detect iLTD.

The developmental loss of iLTD could also result from a reduction in the activity-dependent release of endocannabinoids with age. To examine this possibility, we assessed developmental changes in the response to bath application of the CB1R agonist WIN (10 μM). WIN induced a substantial depression in IPSCs recorded in cells from 3 week-old rats, but not from 5 week-old rats (3 weeks: 73.2±2.4% after 30 min in WIN, n=6,8; 5 weeks 96.6±3.6%, n=8,15, p<0.001. Fig 3A). Since iLTD induction requires both endocannabinoids and presynaptic activity (see Fig 1G), we tested the possibility that baseline activation was insufficient to induce iLTD in the older slices. However, even when paired with a much stronger stimulation (TBS), bath application of WIN did not depress the IPSC amplitude in cells from 5 week-old rats (Fig. 3A). Next, we examined the role of visual deprivation on the developmental loss of endocannabinoid sensitivity. We compared the effects of WIN on rats reared in the dark from weeks 3 to 5 (3N2D; which preserves iLTD), with those on similarly dark reared rats re-exposed to light for 2 days (3N2D+L, which eliminates iLTD). WIN depressed the IPSCs in cells from 3N2D rats (77.3±5.7%, n=4,7. p=0.002. Fig. 3B), but not from 3N2D+L rats (98.5±4.3%, n=5,7. p=0.65), demonstrating a co-regulation of iLTD and the response to the CB1R agonist by age and visual experience.

Figure 3
Sensitivity of IPSC to CB1R agonists is regulated by age and experience

Next we considered the possibility that a developmental increase in the tonic level of endocannabinoids could occlude the effects of exogenous agonist. Such a mechanism might mediate the developmental loss of iLTD in hippocampal CA1, where the CB1R antagonist AM251 increases GABAergic responses in slices from older animals (Kang-Park et al., 2007). However, in layer II/III pyramidal cells from the visual cortex of 5 week-old rats, bath application of AM251 (10μM) did not change IPSC amplitudes (104.4±6.8% at 30 min after AM251, p=0.78. Fig. 3C). In addition, the developmental loss of responsiveness to applied CB1R agonists is restricted to the IPSCs. As shown in Figure 3D, bath application of WIN reduced the glutamatergic EPSC to a similar degree in pyramidal cells from both 3 and 5 week-old rats (3 weeks: 81.8±5.0% at 30 min, n=4,8; 5 weeks: 81.7±6.4, n=3,7; p=0.980). Finally, we evaluated the possibility that changes in CB1 receptors contribute to differences in the responsiveness to endocannabinoids. Quantitative western blot analysis indicated that the CB1R protein content in the visual cortex is higher in 5 week-old rats reared in the dark for two weeks than in age matched normal reared rats (5N: 100.0±3.9%, n=13 rats; 3N2D: 122.3±5.5%, n=15. p=0.004. Fig. S3). Altogether, the results support the idea that the critical period for iLTD induction is mediated by a loss of responsiveness to endocannabinoids, not a change in the supply of endocannabinoids.

Systemic injection of CB1R agonists and antagonists respectively mimics and prevent the effects of visual experience on iLTD

Dark rearing arrests the closure of the critical period for iLTD, while re-exposure to light rapidly stimulates the loss of iLTD (Fig 2). We hypothesized that activity in the visual cortex during light re-exposure releases endocannabinoids, inducing iLTD in vivo and occluding the subsequent induction of iLTD in vitro. Therefore, we tested whether systemic administration of a CB1R agonist during dark rearing occludes the subsequent induction of iLTD in vitro. Experiments were performed in 5 week-old rats that received 3 weeks of normal rearing followed by 2 weeks of dark rearing (3N2D), which express robust iLTD. As shown in Figure 4A, injections of the CB1 agonist WIN (5mg/kg, i.p., twice a day) for 3 days blocked the subsequent induction of iLTD in visual cortical slices (iLTD vehicle-injected controls: 81.6±1.9%, n=7,19; WIN-injected: 99.0±1.2%, n=8,19; t-test: p<0.001). Pre-treatment with the CB1R agonist also depresses PPD to the level seen following iLTD, and occluded a subsequent decrease in PPD following TBS (Figure 4B, PPD in control: 0.20±0.02 pre-TBS and 0.10±0.2 post-TBS, p<0.001; PPD in WIN-treated: 0.14±.0.02 pre-TBS and 0.12±.02 post-TBS, p=0.119). These results indicate that CB1R agonists, like brief light exposure, can reverse the effects of dark rearing and occlude iLTD. We were concerned that residual WIN in the tissue at the time of the experiment might have occluded the induction of iLTD in vitro. Although WIN is rapidly metabolized, with a half-life of about 3 hours in rodents, we tested this possibility by starting the 3-day WIN injection 5 days prior to the experiments, allowing 2 days for clearance of the drug in the dark. The occlusion of iLTD by WIN persisted following this delivery schedule (WIN-injected: 99.7±3.6%, n=6,14; control vehicle injected: 78.0±4.5%; n=5,8; p=0.002), indicating that incomplete clearance of WIN did not occlude the induction of iLTD. These findings are consistent with the idea that dark rearing preserves iLTD in vitro because neural activity in deprived cortex is insufficient to trigger iLTD in vivo.

Figure 4
Endocannabinoid signaling mediates the effects of visual experience on iLTD. Systemic injection of CB1R agonist (WIN: 5mg/kg, i.p., twice a day for 3 days) prevents preservation of iLTD (A) and the TBS-induced decrease in PPD (B) typically seen in 5 week-old ...

Next we tested whether blocking endocannabinoid signaling in vivo prevents the loss of iLTD triggered by 2-days light exposure in 3N2D subjects by injecting the antagonist AM251 for 3 days (5mg/kg i.p. twice a day) starting one day before the light exposure. As shown in Figure 4C, iLTD was robust in cells from light exposed rats injected with AM251 and negligible in cells from control rats injected with vehicle (AM251: 80.35±2.5%%, n=8,18, control vehicle: 99.6±0.6%, n=6,11; p<0.001). The “preserved” iLTD is likely mediated by endocannabinoids as it was blocked by bath applied AM251 (DMSO control: 83.6±1.6, n=3, 6; AM251:102:9±2.3, n=3, 6; p<0.001. Data not shown). 3-day AM251 treatment does not restore iLTD in slices from 5 week-old normal reared rats (107±3.3%, n=4,7,p=0.26. data not shown), indicating that the CB1R antagonist alone does not promote iLTD. Finally, we found that TBS changed PPD in cells from AM251-injected 3N2D+L rats (P<0.001) but not in cells from vehicle injected 3N2D+L controls (p=0.229. Fig 4D). These results indicate that injection of AM251 blocks loss of iLTD induced by light exposure of dark-reared rats, supporting the hypothesis that visually induced cortical activity induces an endocannabinoid-dependent iLTD in visual cortex in vivo.

iLTD mediates the maturation of short-term plasticity

The effectiveness of GABAergic transmission during prolonged stimulation is regulated by short-term synaptic plasticity. IPSCs recorded in layer II/III typically exhibit a short–term depression (STD) evident by a decrease in the amplitude of the IPSC observed during repetitive stimulation until the arrival of a steady state, which depends on stimulation frequency, the release probability and the rate of recovery. STD in visual cortex shares many characteristics with iLTD, including a decrease with age and attenuation of the decrease by dark-rearing from birth (Jiang et al., 2005; Tang et al., 2007). Therefore, we examined whether STD is co-regulated with iLTD by late dark rearing and light exposure. We probed STD with 20 stimulation pulses at 30 Hz because the effects of age and experience on STD are prominent at this frequency (Jiang et al., 2005; Tang et al., 2007). STD was quantified as the amplitude of the IPSC during the steady state (average of the last 5 responses) normalized to the first response. STD was larger in neurons from 5 week-old rats reared in the dark for 2 weeks (3N2D) than in 5 week-old normal-reared rats (5N), or dark-reared re-exposed to light for two days (3N2D+L; Figure 5A,D,E,). Thus, late dark rearing can preserve high levels of STD, which in turn can be reduced by visual experience.

Figure 5
Endocannabinoids, age and visual experience cause parallel reduction in the short-term depression of inhibition

We tested whether we can mimic the developmental reduction in STD by inducing iLTD or WIN application in normal-reared 3 week-old rats. Consistent with induction of iLTD, TBS reduced the response to the first stimulus in the train (69.1±5.2%, p=0.01, n=10, Fig 5B), but did not affect the steady state response (0.327±0.035 of the first pulse before TBS; 0.32±0.029 after TBS, p=0.72). As a result, the relative depression of responses (i.e. STD) was reduced after TBS (p<0.0001 versus non-TBS controls). Similarly, a brief application of WIN (10 min, 10 μM, chased with the antagonist AM251, Fig 5C) reduced the response to the first stimulus in the train (67.7±7.1%, n=11.p<0.001), but had no effect on the steady state response (control: 0.400±0.015; after WIN: 0.388±0.018. p=0.510) and therefore reduced the relative attenuation of the IPSC (p<0.0001 versus DMSO controls). Interestingly, after TBS or WIN was applied to cells from 3 week old rats, the profile of the progressive IPSC attenuation resembled naïve cells in older (5 week-old) rats (Fig. 5D). The TBS- and WIN-induced changes in STD were also comparable to the changes in STD induced by light exposure in dark reared rats (Fig. 5D). As summarized in Figure 5E, the average relative amplitude of the steady state IPSC was equally affected by age (3 weeks: 0.37±0.01,n=7,15; 5 weeks: 0.49±0.02, n=5,15; p<0.001), visual experience (3N2D: 0.35±0.04, n=5,9; 3N2D+L: 0.47±0.004, n=5,12, p=0.02), activity (control: 0.35±0.02; after TBS: 0.47±0.04, n=4,11. p=0.007) and the CB1R agonist (control: 0.39±0.01; after WIN: 0.5 ±0.00; n=5,10. p<0.001). We also examined the effects of age and the CB1R agonist on IPSCs evoked by long and irregular stimulation (160 pulses, with Poisson-distributed inter-stimulus intervals, mean frequency: 30 Hz). Under such regimes, IPSCs are variable because synapses are partially depleted of resources (i.e. synaptic vesicles) and the time for recovering resources varies in each trial. Therefore a reduction in release probability induced by age or CB1R agonists is predicted to reduce vesicle depletion and variability in IPSC amplitude. The results (Fig. S4) indicated that the average relative steady state IPSC amplitude was increased by both age (p=0.002) and bath applied WIN (paired t-test: p=0.011), whereas the coefficient of variation was reduced by both age (p<0.001) and WIN (paired t-test: p=0.009).

Next, we examined how the absence of CB1 signaling would affect the maturation of short-term depression. We found that injection of the CB1R antagonist AM251 prevents the reduction in STP that is normally observed when dark-reared rats are exposed to light (Veh: 0.37±0.02,n=3,26; AM251: 0.46±0.1, n=3,27. p<0.001. Fig 5F,G). In addition, we found that CB1RKO mice do not exhibit a developmental reduction in STD between 3 and 5 weeks of age (WT at 3 weeks: 0.37±0.02, n=3,20;WT at 5 weeks:0.45±0.02,n=4,19; KO at 3weeks: 0.37±0.2,n=6,29; KO at 5weeks: 0.38±0.02, n=5,46. ANOVA: p=0.040. Fig 5F,G). Altogether the results indicate that endocannabinoid activation mimics the effects of age and experience on short-term plasticity at inhibitory synapses, and support the idea the induction of iLTD in vivo is required for the maturation of the properties that govern GABAergic release.

Developmental loss of sensitivity to endocannabinoids in synapses made by fast-spiking interneurons

GABAergic interneurons form a widely diverse group in terms of anatomy, functional properties and molecular composition. To date, CB1 receptors have been detected primarily in CCK-positive interneurons (Katona et al., 1999), and also in somatostatin-positive interneurons (Hill et al., 2007), but not in fast-spiking parvalbumin-positive FS- PV interneurons. However, FS-PV neurons comprise about half of the GABAergic cells in visual cortex (Gonchar and Burkhalter, 1997), provide the bulk of perisomatic inputs, and provide a prominent component of extracellularly evoked IPSCs (Kruglikov and Rudy, 2008; Morales et al., 2002). The high prevalence of iLTD in extracellularly evoked IPSCs (> 70% of cases, see Fig 1D) prompted us to re-examine the possibility that endocannabinoids modulates the synapses made by FS-PV onto pyramidal cells (FS-PV →pyr)

To discriminate between inputs from different interneurons we used Conotoxin (Ctx: 250–300nM) to block the N-type voltage-dependent Ca2+ channels that mediate release in non-FS interneurons, and Agatoxin (Agtx: 500nN) to block the P/Q-type voltage-dependent Ca2+ channels that mediate neurotransmitter release in FS-PV cells, at least in hippocamopus and other neocortical regions (Kruglikov and Rudy, 2008; Wilson et al., 2001; Zaitsev et al., 2007). Each toxin suppressed a complementary fraction of the evoked IPSC in cells from 3 and 5week-old rats (IPSC at 3 weeks in Ctx: 71.3±3.8% of baseline amplitude 20 min after perfusion, n=11,22; in Agtx: 24±7%, n=4,7. At 5 weeks in Ctx: 76.3±1.2%, n=4,7; in Agtx:18.1±5.6, n=4,7, two-way ANOVA: p<0.001. Fig 6A). However, iLTD was induced in each of the toxin-resistant fractions of the IPSC fraction only in cells from 3 week-old, but not from 5 week old rats (iLTD at 3 weeks in Ctxn: 79.8±2.4% of pre-TBS baseline, p<0.001; in Agtx: 68.6±10.1%, p=0.002. At 5 weeks in Ctx: 100.4±2.2% p=0.4; in Agtx127.0±16.8%, p=0.18. Two-way ANOVA: p<0.001). These results suggest that inputs from both FS-PV and nonFS-PV interneuron express developmentally regulated iLTD.

Figure 6
Developmentally regulated iLTD at multiple GABAergic synapses

We also explored whether the developmental loss of iLTD is restricted to inputs recruited with layer IV stimulation. Layer IV and layer II/III stimulation recruit from the same population of GABAergic inputs (Choi et al., 2002; Morales et al., 2002). Therefore, as predicted we found that IPSCs recruited by stimulation of layer II/III also exhibit a loss of TBS-induced iLTD between 3 and 5 weeks of age (iLTD at 3 weeks: 73.6±3.5%, n=5,11; at 5 weeks: 105.7±3.2%. p<0.001. Fig 6B ). In addition we examined developmental changes in the response to application of the CB1R agonist WIN on spontaneously occurring IPSCs (sIPSC), which likely reflect activation of FS-PV since connotoxin did not affect the rate (control: 5.4±1.0; in ctx: 5.3±1.3 Hz; n=2,4. p=0.86, data not shown) or the amplitude (control: 91.1±12.8 pA; in ctx: 84.2±10.4 pA, p=0.31. data not shown). Bath applied WIN (10μM, 15 min) significantly reduced the sIPSC amplitudes in cells from 3 week-old (3W control: 88.6±8.9 pA; WIN: 65.0±4.4; n=6,9. p=0.006 ), has little effect on cells from 5 week-old rats (5W, control:74.6±9.2 pA; WIN: 72.8±10.0,. n=7,9. p=0.29), and reduced the amplitude in cells from 5 week-old rat dark reared for two weeks (NRDR, 103.1±8.9 pA; WIN: 78.4±6.1; n=5,10. p=0.001). The sIPSC frequency was not affected by WIN (3W: p=0.19; 5W: p=0.1; NRDR: p=0.89). These results indicate that the developmental loss of iLTD is not restricted to inputs activated by layer IV stimulation.

The possible endocannabinoid modulation of release in FS-PV cells was intriguing. Therefore, we performed paired recordings in slices prepared from transgenic mice that express GFP in PV-positive interneurons (Goldberg et al., 2005) as model to study unitary IPSCs (uIPSCs) in connected FS-PV→pyr pairs (Fig 7A). We confirmed that the developmental decrease in PPD and CV that we observed in Long Evans rats was also present in the PV-GFP mice (PPD at 2 weeks: 0.36 ± 0.02, n=40,68; PPD at 5 weeks 0.23±0.02, n=25,59. p=0.016, Figure 7D. CV at 2 weeks: 0.56±0.04; CV at 5 weeks: 0.44±0.03, p=0.004). As predicted, bath application of the CB1R agonist WIN (10 μM) modulates the uIPSC’s of FS-PV→pyr in an age- and experience-dependent manner (Fig 7B). In cells from 2 week-old mice WIN induced a rapid and stable depression of the uIPSCs (55.4±0.9% measured at 25–30 min after wash-in, n=13,14). WIN-induced depression of the uIPSCs did not occur when WIN was applied in the presence of the CB1R antagonist AM251 (10 μM, 98.2±3.9%, n=9,10. p=0.52, data not shown) or when the WIN concentration was reduced to 1 μM (1 μM: 91.2±9.6%, 10 μM 40.2±5/8 %, n=4. p=0.013, data not shown). In contrast, WIN did not affect uIPSCs recorded from 5 week-old normally reared mice (94.6%, n=11,14, open triangles in Fig 7B), but induced a significant depression in uIPSCs in slices from 5-week old animals reared in dark for two weeks (57.1±2.1%, n=4,9; filled circles in Fig 7B, p<0.001). Thus, like the extracellularly evoked compound IPSCs, uIPSCs from FS-PV→pyr pairs were modulated by CB1R agonists in an age- and experience-dependent manner.

Figure 7
A critical period for endocannabinoid modulation of synapses between PV fast-spiking interneurons and pyramidal cells in layer II/III of mouse visual cortex

The CB1R-mediated depression of the uIPSCs recorded from FS-PV→pyr pairs was similar to CB1R-mediated depression of the extracellularly evoked IPSCs in additional important aspects. First, depression of the uIPSCs persisted when WIN was applied for 10 min and chased with 10 μM AM251 (67.4±1.2%, n=6,11; p<0.001, Fig 7C). Second, in all experimental groups that responded to WIN, the depression of the uIPSC was associated with a significant (p<0.001) decrease in PPD (Fig 7D), consistent with a reduced probability of release. We further examined the locus of the depression by performing a CV analysis of the uIPSC amplitude. Assuming a binomial model of release, theory predicts that if the mechanism of plasticity has a postsynaptic locus, the changes in CV2 should be constant; if the mechanism has a presynaptic locus, the changes in CV2 should be ≥ the changes in the mean (Faber and Korn, 1991; Sjostrom et al., 2003). In a plot of the normalized 1/CV2 vs normalized changes in the mean amplitude of uIPSCs, the data points locate on or under the diagonal (Fig 7E), consistent with a presynaptic locus.

Finally, we checked the modulation of the FS-PV→pyr connections in slices prepared from the rat visual cortex to confirm that the data obtained from the transgenic mice are consistent with those obtained from the rats. In rats the FS-PV interneurons were identified electrophysiologically with firing rates higher than 100 Hz and thin action potentials. We found that bath application of WIN (10 μM, 10 min) induced a lasting depression of uIPSCs (60.8±6.0%, n=11,14; p<0.001) that was associated with a decrease in paired-pulse depression (p<0.001) and increases in the CV2 (Suppl. Fig 3). Altogether, the results obtained with paired recordings suggest that CB1R-mediated iLTD regulated the inputs of FS-PV inputs onto layer II/III pyramidal cells.

DISCUSSION

In GABAergic synapses in layer II/III of the immature visual cortex the probability of release is high, resulting in pronounced short-term depression (Jiang et al., 2005; Tang et al., 2007). Here we present evidence that the transition to a mature state with low release probability and reduced STD is an experience-dependent process that requires the recruitment of endocannabinoid-dependent mechanisms similar to those that subserve iLTD in vitro. In support of this proposition we demonstrate that 1) endocannabinoid-dependent presynaptic iLTD is expressed in pyramidal neurons of layer II/III only during a critical period, which can be arrested but not reversed by visual deprivation, 2) synapses made by fast-spiking interneurons exhibit developmentally constrained iLTD and 3) although iLTD reduces the probability of GABA release, it also reduces short-term depression and the variability of synaptic transmission during irregular patterns of stimulation, similar to the changes we observe in response to visual experience. Together this suggests a novel role of endocannabinoid-mediated synaptic plasticity in the maturation of GABAergic transmission in the mammalian visual cortex, which may serve to shift the relative efficacy of GABAergic transmission to higher frequencies.

Endocannabinoid-dependent forms of presynaptic LTD have been described at both excitatory and inhibitory synapses in multiple brain regions, and a diversity of mechanisms appears to link synaptic activity to endocannabinoid synthesis and LTD induction (Chevaleyre et al., 2006; Hashimotodani et al., 2007; Lovinger, 2008). The rules of induction of iLTD in layer II/III pyramidal neurons appear to be similar to those described in hippocampal CA1 neurons (Chevaleyre and Castillo, 2003), although we have not identified which endocannabinoid species is involved in layer II/III iLTD. In both cases iLTD is only induced in active GABAergic inputs, and glutamatergic release is required to activate mGluR5 receptors and PLC, suggesting a heterosynaptic induction mechanism. However, there are seemingly mechanistic differences: CA1 iLTD requires spontaneous firing in the interneurons to activate calcineurin (Heifets et al., 2008) in the axon terminals, whereas FS(PV) cells rarely fire in cortical slices (Maffei et al., 2004). It is unclear whether in visual cortex calcineurin is not necessary, or if it is sufficiently activated by baseline stimulation alone.

The mechanisms for the developmental loss of iLTD are more difficult to compare due the scarcity of data. Prior to our study, developmental changes in endocannabinoid-mediated LTD have only been reported in inhibitory synapses in CA1 (Corlew et al., 2007; Kang-Park et al., 2007) and in excitatory synapses in layer II/III of visual cortex (Huang et al., 2008). The developmental reduction of iLTD in CA1 has been attributed to an increase in the tonic level of endogenous agonists around puberty (Kang-Park et al., 2007). In contrast, we demonstrate that the developmental loss of iLTD in layer II/III pyramidal neurons is associated with the loss of response to endocannabinoid agonists. In FS-PV→pyr synapses, this loss of responsiveness coincides with the end of synaptogenesis. The relationship between synaptogenesis and iLTD is unclear, especially given that at this stage visual experience drives many unrelated changes in synaptic function. Nevertheless, a simple scenario to consider is that iLTD is induced in vivo by visually evoked activity and somehow drives the loss of responsiveness to endocannabinoids. The target of CB1R agonists would therefore be only naïve synapses; consequently, the response to endocannabinoids disappears with the end of GABAergic synaptogenesis. Whether the CB1Rs remain in FS(PV) axon terminals after the end of the critical period, and whether iLTD becomes irreversible once induced remain open questions.

Our study is the first to report endocannabinoid modulation of synaptic function made by FS-PV interneurons in the mammalian cortex, although such modulation has been documented in the amygdala (McDonald and Mascagni, 2001) and basal ganglia (Freiman et al., 2006; Narushima et al., 2006; Uchigashima et al., 2007). In cortex it was reported that FS-PV neurons were immunocytochemically negative for CB1 receptors (Katona et al., 1999). However, it is possible that immunocytochemistry is not sensitive enough to detect CB1Rs in PV-positive interneurons. There are several examples in which immunocytochemical methods did not detect the presence of CB1Rs, but cannabinoid modulation was confirmed with other methods (Hill et al., 2007; Sjostrom et al., 2003). Our finding that WIN does not affect the FS(PV)-mediated uIPSC at 1 μM but it depresses them at 10 μM suggest a low content of CB1 receptors in PV containing axon terminals. A relative lower CB1 content at the PV-axon terminal could also explain why 1 μM WIN depresses the CCK-mediated, but not the FS(PV)-mediated uIPSC (Galarreta et al., 2008). Alternatively, higher concentrations of WIN might recruit additional processes (other than CB1Rs) that are also required for this form of iLTD. Regardless of the mechanism, the requirement for a high dose of agonist predicts that the threshold for iLTD induction would be rarely achieved in vivo in the absence of visual experience.

Recent work has shown that presynaptic forms of iLTD and iLTP coexist at the same synapses (Nugent et al., 2007; Pan et al., 2008). Postsynaptic form of LTP and LTD are generally regarded as activity-dependent mechanisms that allow bi-directional modification of synaptic strength. Interestingly, the reduction in inhibitory function after iLTD in CA1 pyramidal neurons also lowers the threshold for activity-dependent postsynaptic LTP at excitatory synapses (Chevaleyre and Castillo, 2004). Here we demonstrate that iLTD plays additional roles in the visual cortex. Our finding that iLTD reduces the responses evoked at low frequencies (0.03 to 0.05 Hz) but not at high frequencies (30 Hz) (Fig 5B) is consistent with a redistribution model of presynaptic plasticity in which changes in synaptic strength depend on the stimulation frequency (Abbott et al., 1997; Tsodyks and Markram, 1997). Thus, iLTD might enhance rather than reduce GABAergic function during high firing rates. One proposed role of fast-spiking interneurons is to integrate network activity and to broadcast an inhibitory signal that confers homeostatic stability to the network. Such a negative feedback function would be better served by synapses with low probability of release that attenuate less during sustained firing than by synapses with high probability of release that rapidly deplete synaptic resources.

It is interesting to note that in parallel with the developmental decrease in the probability of release, fast-spiking GABAergic cells also increase the expression of potassium channels responsible for the repolarization of the action potential (Goldberg et al., 2005). This shortens the duration of the action potential, thereby reducing the probability of the release per single action potential, but allows higher rates of firing and release. This suggests a developmental orchestration of mechanisms aimed at enhancing GABAergic performance during high frequency activation, at the expense of low frequency performance. During maturation, the reduction in the probability of release at low frequencies may be compensated by the 2 to 3-fold increase in the number of release sites. Whether such a developmental increase in the capacity fast transmission contributes to the closure of critical period remains to be determined. Finally, the principles we describe for the maturation of GABAergic inputs to layer II/III pyramidal cells might be more general. In all cases examined, glutamatergic synapses in sensory cortices undergo a developmental reduction in paired-pulse depression (Feldmeyer and Radnikow, 2009; Frick et al., 2007; Reyes and Sakmann, 1999; Yanagisawa et al., 2004) and also deprivation-induced increases in short-term depression (Finnerty et al., 1999), similar to what we describe for GABAergic synapses. It is possible that endocannabinoid-mediated regulation of synaptic transmission may be a ubiquitous mechanism in the developmental maturation of synaptic transmission at both GABAergic and glutamatergic synapses.

EXPERIMENTAL PROCEDURES

Visual cortical slices (300 μm) from Long-Evans rats and C57BL/6 mice reared in normal light/dark 12 hour cycles, or in the dark for defined periods with care provided under infrared illumination. Slices were cut as described (Kirkwood and Bear, 1994) in ice-cold dissection buffer containing (in mM): 212.7 sucrose, 5 KCl, 1.25 NaH2PO4, 10 MgCl2, 0.5 CaCl2, 26 NaHCO3, 10 dextrose, bubbled with 95% O2/5% CO2 (pH 7.4). Slices were transferred to normal artificial cerebrospinal fluid (ACSF) for at least an hour prior to recording. Normal ACSF was similar to the dissection buffer except that sucrose was replaced by 124 mM NaCl, MgCl2 was lowered to 1 mM, and CaCl2 was raised to 2 mM.

Visualized whole-cell voltage-clamp recordings were made from layer II/III pyramidal glass pipettes filled with intracellular solution containing (in mM: CsCl 140, CaCl2 0.2, NaCl 8, EGTA 2, NaGTP 0.5, MgATP 4, and HEPES 10, pH 7.2). Only cells with membrane potentials < −65 mV, series resistance <20 MΩ, and input resistance >100 MΩ were studied. Cells were excluded if input resistance changed > 15% over the experiment. Data were filtered at 5 kHz and digitized at 10 kHz using Igor Pro (WaveMetrics Inc., Lake Oswego, Oregon). Synaptic currents were recorded at −60 mV in the presence 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μM 2-amino-5-phosphonovaleric acid (APV), and evoked every 20 sec by stimulating layer IV with 0.2 ms pulses delivered in pairs (inter-stimulus interval: i.s.i= 100 msec) to compute paired-pulse depression (PPD=1-p2/p1, where p1 and p2 are the amplitude of the response to the first and second stimulation, respectively). Stimulation was delivered through concentric bipolar stimulating electrodes (FHC, Bowdoin, ME) with intensity adjusted to evoke 100–300 pA responses. In dual-cell recordings, the interneuron was recorded under current clamp with a pipette containing (in mM): 130 K-gluconate, 10 KCl, 10 HEPES, 0.5 Na3GTP4, MgATP and 10 Na-phosphocreatine. Fast-spiking cells were identified under fluorescence, in the case of the GFP mouse line, or by their fast spiking capabilities. Synaptic strength was quantified as the IPSC amplitude and 10 min of stable baseline (less than 10% change) was allowed before any manipulation. ILTD was induced with theta burst stimulation (TBS), consisting on 4 theta burst epochs delivered at 0.1 Hz. Each TBS epoch consisted of 10 trains of 4 pulses (100 Hz) delivered at 5 Hz. See supplementary methods for details of the analysis of spontaneous IPSCs

Drug solutions

: For systematic injection of CB1 receptor antagonist and agonist, AM251[1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide] and WIN55,212-2 {(R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone} were dissolved in vehicle (composed of 10% Tween 80, 20% DMSO and 70% saline (0.9% NaCl)) to a final concentration of 1mg/mL. Each rat was intraperitoneally injected (5mg/kg) twice a day (12 h interval) for 3 days. For in vitro experiments, AM251 or WIN were dissolved in DMSO in stock solution and diluted it just before use in ACSF to the indicated final concentration. AM251, Agatoxin, WIN, U-73122 and MPEP were purchased from Tocris; CNQX, APV, Tween 80, ω-Conotoxin and DMSO from Sigma.

Supplementary Material

01

Acknowledgments

We thank Dr. E Quinlan for insightful comments, Drs J. Pickel and J.Z. Huang for the CB1R KO and the GFP(FS) mice, and Dr K Mackey for the CB1R antibody. Supported by RO1 EY12124.

Footnotes

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