Authors: Cesar C. Ceballos, Lei Ma, Maozhen Qin, Haining Zhong
Categories: Article, Cellular neuroscience, Synaptic vesicle exocytosis
Source: Communications Biology
Several brain neuronal populations transmit both the excitatory and inhibitory neurotransmitters, glutamate, and GABA. However, it remains largely unknown whether these opposing neurotransmitters are co-released simultaneously or are independently transmitted at different times and locations. By recording from acute mouse brain slices, we observed biphasic miniature postsynaptic currents, i.e., minis with time-locked excitatory and inhibitory currents, in striatal spiny projection neurons. This observation cannot be explained by accidental coincidence of monophasic excitatory and inhibitory minis. Interestingly, these biphasic minis could either be an excitatory current leading an inhibitory current or vice versa. Deletion of dopaminergic neurons did not eliminate biphasic minis, indicating that they originate from another source. Importantly, we found that both types of biphasic minis were present in multiple striatal neuronal types and in nine out of ten other brain regions. Overall, co-release of glutamate and GABA appears to be a widespread mode of neurotransmission in the brain.
**Subject ** Synaptic vesicle exocytosis, Cellular neuroscience
The long-accepted Dale’s principle postulated that each neuron releases a single transmitter type^1,2^. However, recent works have demonstrated that certain neuronal populations are capable of releasing two or more neurotransmitters^3–5^. There are two qualitatively distinct modes of releasing multiple neurotransmitters from the same neurons. They can be simply “co-transmitted”, i.e., two transmitters being released from different vesicles at different locations and/or times. Alternatively, they can be “co-released” simultaneously at the same location, typically from the same vesicle. These two modes of release have distinct implications for how the two neurotransmitters coordinate with each other for function^3,4^.
Among the multimodal neurons, a peculiar subset can co-transmit glutamate and GABA, the primary excitatory and inhibitory neurotransmitters in the brain, respectively^6–16^. How these two neurotransmitters are released is being intensively studied. Given the opposing functions of these two neurotransmitters, it would seem logical if they are released from different synaptic pools. However, recent evidence at the lateral habenula suggest that glutamate and GABA can be co-released from the same synaptic vesicles^16,17^. Furthermore, single-cell RNA sequencing and immunostaining studies suggest that vesicular transporters for both glutamate and GABA (VGAT and VGLUT1/2/3, respectively) are co-expressed in many neuronal populations throughout the brain, sometimes even on the same synaptic vesicles^18–24^. Nevertheless, it remains unclear whether co-release is a general mechanism for glutamate/GABA bimodal neurons and whether such co-release occurs broadly in the brain.
We set out to answer these questions first in the dorsolateral striatum. It is well established that dopaminergic axon terminals from substantia nigra pars compacta (SNc) can release glutamate and GABA in addition to dopamine in the striatum^6,25–29^, but whether these neurotransmitters are co-released is still under investigations^9,30–35^. We recorded miniature postsynaptic currents (minis), which are thought to arise from the release of neurotransmitters by a single vesicle. At a holding voltage that can detect both excitatory and inhibitory synaptic currents, we observed “biphasic” minis in striatal projection neurons (SPNs) that depended on both AMPA and GABAA receptors and occurred beyond the coincidence chance expected of independent excitatory and inhibitory minis. The frequency of biphasic minis could also be modulated independently from those of monophasic minis. Minimal optogenetic stimulation experiments and neuronal ablation experiments demonstrated that these biphasic minis did not depend on midbrain dopaminergic neurons. Surprisingly, such biphasic minis were detected in all but one of 12 brain regions or cell types that we have examined. Our results suggest that glutamate and GABA co-release is a widespread feature of a subset of synapses throughout the brain.
Biphasic miniature synaptic currents, mediated by both AMPA and GABAA receptors may indicate co-release from the same synaptic vesicles^16,36–38^. To determine whether such co-release occurs in the dorsolateral striatum, we recorded minis from putative SPNs in acute brain slices of wildtype mice using whole-cell patch-clamp recording in the presence of TTX (1 µM). Both miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively) were observed in the same trace when the cell was held at -30 mV, but less so at -10 or -50 mV (Figs. 1a, b and Supplementary Fig. 1). Careful visual examination of the traces at -30 mV revealed a unique type of mini that exhibited a clear biphasic shape (Fig. 1c–e). Nearly all of these minis (181 out of 184) were well fit by an averaged mEPSC followed by a mIPSC with a small delay (Fig. 1e; delay = 9.1 ± 3.9 ms, mean ± s.d.). Similar observations were found using a semi-automatic template-matching approach (Supplementary Fig. 2a and b). These biphasic minis were blocked by either the AMPA receptor (AMPAR) antagonist NBQX (10 µM) or the GABAA receptor (GABAAR) antagonist GABAzine (10 µM) (Fig. 1f, g). As the holding voltage was moved from -50 to -10 mV, the inward component decreased, and the outward component increased (Fig. 1h, i). These results indicate that the biphasic minis are the result of nearly simultaneous activation of AMPA and GABAA receptors.
Fig. 1 Detection of straight biphasic minis in SPNs of the dorsolateral striatum.a Example traces of miniature current recordings at different holding potentials (V
H). b Example traces of a mIPSC (top) and a mEPSC (bottom). c Example traces of straight biphasic minis at VH= −30 mV. d Frequency (# events/min) of biphasic minis, mEPSCs and mIPSCs at VH= −30 mV. e Histogram of the onset delay between the mEPSC and mIPSC component used to fit the biphasic minis. Mean ± s.d. = 9.1 ± 3.9 ms. Inset, biphasic mini fit by a 7-ms delay. For panels c–e n = 181 events, 11 neurons, and 4 mice. f, g Biphasic minis are abolished by either AMPAR (f) or GABAAR (g) antagonists (NBQX or GABAzine, respectively). The scale bars are applied to both panels. n (neurons/slices/mice) = 5/5/3 for both. Two-sided paired t-test on normalized data (to control). From top to bottom (except controls), p = 1.4 × 10^−5^, 0, 0, and 0; Cohen’s d = 3.71, 3.06, 0.87, and 0.87. h Average traces of biphasic minis at different holding potentials. i Voltage-dependent changes of the amplitude of the maximum and minimum peaks of the straight biphasic minis. j The probability of observed straight biphasic minis normalized to the probability of their occurrence due to coincidence of independent mEPSC and mIPSC (two-sided paired t-test on the logarithmic scale; p = 1.3 × 10^−8^; Cohen’s d = 2.77). n (neurons/slices/mice) = 11/7/4 for panels h–j. ***p ≤ 0.001. All error bars represent s.e.m.
We asked whether biphasic minis were caused by coincident arrival of independent monophasic mEPSCs and mIPSCs. All mini events followed Poisson distributions (Supplementary Fig. 3), indicating that individual events are independent from each other. We then calculated the likelihood of mEPSCs and mIPSCs arriving in the same time window (10 ms; see Fig. 1e) using the observed mEPSC and mIPSC frequencies (see Methods) and compared this to the probability of biphasic minis from our recordings. The frequency of biphasic minis was significantly lower than either mEPSCs or mIPSCs (Fig. 1d) but was still about 10-fold higher than chance (p < 0.001; Fig. 1j and Supplementary Fig. 2c), suggesting that biphasic minis are the result of correlated release of glutamate and GABA.
Interestingly, in the same recording, we also observed biphasic minis in which the outward current preceded the inward current (Fig. 2a, b). We called this second type “reverse” biphasic minis, whereas the original type was termed “straight”. The reverse biphasic minis were also sensitive to the blockade of either AMPARs or GABAARs (Fig. 2c, d) and were well fit by an mIPSC preceding an mEPSC, with an average delay of 11.1 ± 3.9 ms (mean ± s.d.; Fig. 2e). Reverse biphasic minis also occurred well above the chance of coincident independent mEPSCs and mIPSCs (p < 0.01; Fig. 2f), suggesting that they reflect correlated release of glutamate and GABA.
Fig. 2 Reverse biphasic minis in SPNs of the dorsolateral striatum.a Example traces with reverse biphasic minis (dashed red box). b Example traces of reverse biphasic minis at V
H= −30 mV. c, d Reverse biphasic minis are abolished by NBQX (c) or GABAzine (GBZ; d). n (neurons/slices/mice) = 5/5/3 for both. Two-sided paired t-test on normalized data (to control). From left to right (except controls), p = 0, 0, 0, and 0; Cohen’s d = 1.26, 1.26, 0.81, and 0.81. e Histogram of the onset delay between the mEPSC and mIPSC component used to fit the biphasic minis. n = 151 events. Mean ± s.d. = 11.1 ± 3.9 ms. Inset, a reverse biphasic mini fit by a 6-ms delay. f The probability of observed reverse biphasic minis normalized to the probability of their occurrence due to coincidence of independent mEPSC and mIPSC. n (neurons/slices/mice) = 11/7/4 (two-sided paired t-test on the logarithmic scale, p = 0.0038, Cohen’s d = 0.99). g Example average traces of straight and reverse biphasic minis recorded in dSPNs and putative iSPNs. h, i Frequency (events/min) of straight (h) and reverse biphasic minis (i) from paired dSPNs and iSPNs at VH= −30 mV. n (neurons/slices/mice) = 8/5/2. Two-sided paired t-test. From h, i, p = 0.17 and 0.25; Cohen’s d = 0.55 and −0.45. j–l Example average traces (j), frequency (k), and occurrence probability normalized to chance (l) of straight and reverse biphasic minis recorded in ChIN at VH= −30 mV. n (neurons/slices/mice) = 7/3/2 for straight and 6/3/2 for reverse. Two-sided paired t-test on the logarithmic scale, p = 9 × 10^−6^ and 0.01, Cohen’s d = 2.79 and 1.27. **p ≤ 0.01. ***p ≤ 0.001. All error bars represent s.e.m.
To examine whether biphasic minis may be specific to a unique neuronal type in the striatum, we used the D1R-tdTomato mouse line to distinguish direct pathway SPNs (dSPNs) from putative indirect-pathway SPNs (iSPNs; i.e., tdTomato-negative neurons that were not cholinergic interneurons [ChINs], which have a uniquely large soma). Both types of biphasic minis were present in dSPNs and iSPNs at comparable frequencies that were above chance (Fig. 2g–i). Furthermore, both straight and reverse biphasic minis were also present in ChINs (Fig. 2j–l). Thus, biphasic minis of both types appeared to be broadly present in multiple neuronal types in the striatum.
To further determine whether biphasic minis are independent from monophasic mEPSCs and mIPSCs, we examined how they were modulated by neuromodulators. Specifically, following the lead from a recent study on glutamate/GABA co-release in the lateral habenula^17^, we examined how adenosine and serotonin affected the minis in the striatum. We found that adenosine decreased the frequency but not the amplitude of both mEPSCs and mIPSCs (Fig. 3a–d). The frequencies of both types of biphasic minis were also decreased (Fig. 3e, f). However, when compared to the chance of coincident mEPSCs and mIPSCs, which is related to the multiplicative effect of both mEPSC and mIPSC frequencies, the relative probability of both types of biphasic minis in fact increased (Fig. 3g, h). In contrast to adenosine, serotonin application did not alter the frequencies of minis (Supplementary Fig. 4a–h). Removal of external calcium also did not alter mini frequencies, indicating that biphasic minis are not multivesicular release events triggered by calcium influx (Supplementary Fig. 4i–p). Overall, these results strengthen the notion that biphasic minis are unlikely the result of coincidently arrived mEPSCs and mIPSCs.
Fig. 3 Adenosine decreased the number but not the probability of biphasic minis.a–d Frequency of mEPSCs (a) and mIPSCs (b), and amplitudes of mEPSCs (c) and mIPSCs (d) of SPN neurons before (ctrl) and after the bath application of 20 μM adenosine (Ado). e. f Frequency of straight (e) and reverse (f) biphasic minis before (ctrl) and after adenosine application (p = 0.025, p = 0.015 respectively). Two-sided paired t-test on normalized data (to control). From a–f, p = 1.0 × 10^−6^, 2.2 × 10^−5^, 0.35, 0.81, 0.021, and 6.9 × 10^−4^; Cohen’s d = 1.70, 3.54, 0.39, 0.10, 1.0, and 1.13. g, h The probability of observed straight (g) or reverse biphasic minis (h) normalized to the probability of their occurrence due to coincidence of independent mEPSC and mIPSC in the same cells before (ctrl) and after adenosine application. All recordings were done at −30 mV. n (neurons/slices/mice) = 8/8/3 for both control and adenosine application for panels a–g, and 7/7/3 for panel h. Two-sided paired t-test on the logarithmic scale. For panel g (control and adenosine), p = 5.6 × 10^−5^ and 9.2 × 10^−5^; Cohen’s d = 3.05 and 2.82. For panel h (control and adenosine), p = 0.0084 and 0.0013; Cohen’s d = 1.46 and 2.15. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. All error bars represent s.e.m.
Because dopaminergic axons can release both glutamate and GABA^6,26^, we asked whether they might contribute to the biphasic minis. First, we performed minimal optogenetic stimulations of dopamine axon terminals. A single short pulse of blue light ( ~ 470 nm, 1 ms, ~2.5 mW/mm^2^) was applied to a field (Φ = 440 μm) centered on the recorded SPN in slices prepared from DAT-Cre/Ai32 double heterozygous mice, which express channelrhodopsin (ChR2[H134R]-EYFP) in dopaminergic axons. The light intensity was adjusted so that failure and apparent quantal synaptic responses could be observed in different trials (Fig. 4a). Four types of responses were failures, EPSCs only, IPSCs only, and biphasic PSCs (Fig. 4a, b). However, the probability of biphasic PSCs was not statistically higher than chance in this experiment (p = 0.29, sign test; Fig. 4c).
Fig. 4 Dopaminergic axons are not the major source of biphasic minis.a Example single events observed upon minimal optogenetic stimulation with the same cell (left) and the class average (right). b Percentage of different types of events. n (neurons/slices/mice) = 11/8/3. c Scatter plots of chance versus probability of measured biphasic event frequency (two-sided sign test; p = 0.29; Cohen’s d = −0.55). d, e Unilateral injection of 6-OHDA assessed by tyrosine hydroxylase (TH) immunofluorescence in the striatum after 4 days. f, g Example traces of straight and reverse biphasic minis (f) and their frequency (g) recorded in control and 6-OHDA lesioned hemispheres. n (neurons/slices/mice) = 16/8/4 for ctrl, and 14/7/4 for 6-OHDA. Two-sided Wilcoxon rank sum test. For straight and reverse, p = 0.68 and 0.83; Cohen’s d = 0.071 and 0.14. All recordings were done at −30 mV. All error bars represent s.e.m.
Second, we unilaterally injected 6-hydroxydopamine (6-OHDA) into the SNc to deplete dopaminergic neurons (Fig. 4d). Near complete removal of tyrosine hydroxylase-positive axon terminals in the ipsilateral dorsal striatum was observed after 4 days (Fig. 4e). At day 4 and 5 post injection, comparable frequencies of both straight and reverse biphasic minis were observed between the control and injected hemispheres (Fig. 4f, g; p = 0.68 for straight and 0.83 for reverse, Wilcoxon rank sum test). There were also no differences in frequency or amplitude of monophasic mEPSCs and mIPSCs (Supplementary Fig. 5). Together, these results suggest that dopamine axon terminals are not the main source of either biphasic or monophasic minis in the dorsolateral striatum.
Because our earlier results indicated that biphasic minis were widespread across neuronal types in the striatum, we asked whether they are also widespread across brain regions. In addition to SPNs and ChINs in the dorsolateral striatum, we recorded in the following brain regions of wildtype mice (Fig. 5a and Supplementary Fig. 6): barrel cortex (BC), basolateral amygdala (BLA), CA1 region of hippocampus (CA1), cerebellum (Cb), globus pallidus externa (GPe), medial prefrontal cortex (mPFC), suprachiasmatic nucleus (SCN), substantia nigra pars compacta (SNc), ventral thalamus (Th) and primary visual cortex (V1). Surprisingly, we observed biphasic minis in all regions.
Fig. 5 Biphasic minis are widespread throughout the brain.a Brain regions where electrophysiological recordings were the barrel cortex (BC), basolateral amygdala (BLA), CA1 region of hippocampus (CA1), cerebellum (Cb), cholinergic interneurons from dorsolateral striatum (ChIN), globus pallidus externa (GPe), medial prefrontal cortex (mPFC), spiny projection neurons (SPN) from dorsolateral striatum (STR), suprachiasmatic nucleus (SCN), substantia nigra pars compacta (SNc), ventral thalamus (Th), primary visual cortex (V1). b–d Example of average traces (b), frequency (c), and probability normalized to chance (d) of straight biphasic minis across brain regions and cell types. From left to right in panels c, d n (neurons/slices/mice) = 14/6/3, 9/7/3, 10/4/2, 11/6/3, 7/3/2, 5/4/2, 11/3/3, 16/7/4, 8/3/3, 11/6/3, 6/4/3, and 5/3/2. In panel d two-sided paired t-test on the logarithmic scale. p = 4.2 × 10^−6^, 2.4 × 10^−^^5^, 2.0 × 10^−6^, 0.99, 3.2 × 10^−4^, 0.019, 7.3 × 10^−8^, 2.3 × 10^−8^, 0.017, 0.0018, 0.022, and 0.0023; Cohen’s d = 2.018, 2.89, 3.39, −0.002, 2.79, 1.69, 4.19, 2.65, 1.11, 1.27, 1.35, and 3.11. e–g Example of average traces (e), frequency (f), and probability normalized to chance (g) of reverse biphasic minis across brain regions and cell types. From left to right in panels f and g, n (neurons/slices/mice) = 14/6/3, 8/7/3, 9/4/2, 11/6/3, 6/3/2, 4/4/2, 11/3/3, 16/7/4, 7/3/3, 11/6/3, 6/4/3, and 6/3/2. In panel g, two-sided paired t-test on the logarithmic scale. p = 7.3 × 10^−4^, 0.0074, 7.3 × 10^−5^, 0.043, 0.027, p = 0.56, 5.5 × 10^−5^, 3.8 × 10^−5^, 0.84, 0.78, 0.44, and 0.004; Cohen’s d = 1.17, 1.32, 2.48, −0.69, 1.27, −0.33, 2.01, 1.44, 0.081, 0.086, 0.34, and 2.051. All recordings were done at −30 mV (two-sided paired t-test). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. All error bars represent s.e.m.
Straight biphasic minis occurred at a frequency of ~1–20 events/min that varied across brain regions (Fig. 5b, c). When compared with their chance levels calculated from measured mEPSC and mIPSC frequencies (Supplementary Fig. 7), straight biphasic minis occurred above chance in all regions except the cerebellum (Fig. 5d). Reverse biphasic minis had a frequency of ~1–12 events/min (Fig. 5e, f) and was above chance in seven out of 12 brain regions/cell types (Fig. 5g). In the barrel cortex, we also verified that both types of biphasic minis could be abolished by either NBQX or GABAzine (Supplementary Fig. 8). These results suggest that glutamate/GABA co-release is widespread across brain regions.
Here, we investigated GABA and glutamate co-release using electrophysiological recordings of miniature postsynaptic currents. We found that biphasic minis involving both AMPA and GABAA receptors were widespread throughout many brain regions. Statistical analysis showed that the observed biphasic minis cannot be explained by coincident independent release of GABA and glutamate from separate vesicles.
Although many studies have shown evidence of axons co-transmitting glutamate and GABA in the brain^6–16,23,30,37,39–45^, evidence for glutamate and GABA co-release is scarce with the exception of those from the entopeduncular nucleus (EP) axons onto the lateral habenula^16,17^. We found that eleven out of the 12 tested brain regions or cell types exhibited glutamate/GABA biphasic minis, which are thought to likely represent the co-release of these two opposing transmitters from a single vesicle^3,4,16,36–38,46^. While surprising, this finding is consistent with recent studies showing that a small percentage of synaptic vesicles co-express both glutamate and GABA vesicular transporters in many brain regions^13,18–21,47^. At the postsynaptic side, GABAA and AMPA receptors have been found to colocalize at mossy fiber synapses^48^.
Interestingly, we found two types of biphasic ones in which the mEPSC component leads the mIPSC component (straight biphasic minis) and others with the mIPSC preceding the mEPSC (reverse biphasic minis). To our knowledge, the reverse biphasic mini has not been previously described. We verified that both types of biphasic minis depend on both AMPA and GABAA receptors. Assuming that these biphasic minis arise from single-vesicular releases, the two types of biphasic minis likely reflect differences in synaptic organizations. For example, at synapses giving rise to straight biphasic minis, AMPA receptors may be intermingled with or be more central to the active zone than GABAA receptors, whereas at synapses with reverse biphasic minis, GABAA receptors could be closer to the release site. The delay between the AMPA and GABAA events within a biphasic mini is about 10 ms. This is seemingly long based on the prediction of a synapse with simple receptor distribution and synapse cleft geometry^49^. However, the neurotransmitter diffusion may be slowed down by buffering and potential spatial hindering by astrocytic processes^50,51^, and the non-centrally localized receptors may be positioned outside of the immediate postsynaptic density. While it has been shown that AMPAR and GABAAR may exist in the same synapses or spines^48,52,53^, direct testing of these possibilities will likely require EM tomography study of neuronal synapses. Finally, our data cannot rule out the possibility that glutamate and GABA are packaged into separate vesicles, but the release of these distinct vesicles are tightly coupled within ten milliseconds. Coupled multivesicular release within an active zone triggered by putative single calcium channel opening events or by local calcium sparks has been observed in specialized synapses^54,55^. Another possibility is that neurotransmitter release from one terminal diffuse to an adjacent synapse to increase its release probability within milliseconds^56^. Regardless of the precise mechanism, our results show that coordinated, time-locked co-release of glutamate and GABA is surprisingly common in the brain.
What is the function of glutamate/GABA co-release? First, although biphasic minis account for only a small fraction of total minis, they may be dominant in particular presynaptic neuronal types to shape their function. There have been several suggested possible mechanisms. Colocalization of GABAA receptors with AMPA receptors could rapidly inhibit excitatory responses, providing a fast and more targeted form of ‘surround’ inhibition^57^. Both computer simulations and experimental evidence suggest that inhibition within a few milliseconds and in close proximity to glutamate receptor excitations is highly efficient in attenuating postsynaptic calcium response amplitudes without impacting voltage changes^52,53,58^. The spatial specificity of calcium dynamics may also be enhanced^58^. Additionally, VGLUT expressed in axon terminals may result in enhanced uptake of GABA into synaptic vesicles (i.e., vesicular synergy)^19,36^, thereby increasing GABA release. Finally, there may be potential implications for plasticity. Activation of presynaptic metabotropic glutamate receptors or GABAB receptors could modulate local synaptic transmission^3,13,27,28,59^. Calcium influx through NMDARs have also been found to selectively potentiate inhibition from a subset of inhibitory synapses in somatostatin positive interneurons^60^.
The cellular origin of glutamate/GABA co-release remains unknown. In the striatum, our results suggest that dopaminergic axons are not the major contributor to glutamate and GABA co-release. Although we did not examine the nucleus accumbens where dopaminergic axon’s glutamate release probability may be different^61^, our results are consistent with recent investigations. There appears to be minimal colocalization between VGLUT2 and VMAT2—which is responsible for vesicular GABA transport in these axons^7,42,62^—in dopaminergic axonal terminals in the striatum^31–33,63^ (but see also ref 35). Additionally, glutamate and dopamine release each exhibits different properties^31,40,61,63,64^. One possible source might be GABAergic interneuron axon terminals that also express VGLUT2 or VGLUT3. For instance, GABA neurons of the hypothalamic anteroventral periventricular nucleus express VGLUT2^65^. Likewise multiple populations of GABAergic interneurons in the cerebral cortex and hippocampus^43,66,67^ as well as immature GABAergic synapses from neurons of the medial nucleus of the trapezoid body in the lateral superior olive (LSO)^44^ express VGLUT3. Overall, it appears that neurons that release glutamate and GABA express either VGLUT2 or VGLUT3, but not VGLUT1^45^. Future revelation of the precise cellular source of these biphasic minis will enable targeted manipulation to dissect the function and plasticity of this wide-spread co-release of glutamate and GABA.
Animal handling and experimental protocols were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals, written by the National Research Council (US) Institute for Laboratory Animal Research, and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Oregon Health & Science University (#IP00002274). DAT-IRES-cre (B6.SJL-Slc6a3^tm1.1(cre)Bkmn^/J; Jax #006660) homozygous and Ai32 (B6;129S-Gt(ROSA)26Sor^tm32(CAG-COP4H134R/EYFP)Hze*^/J; Jax #012569) homozygous mice were bred for minimal optogenetic stimulation experiments. D1-Tdtomato (Jax #016204) hemizygous mice were bred with C57BL/6NCrl mice. For crossing, both reciprocal crosses were used. For experiments, both sexes of mice ~ P45 (mean ± s.e.m. = 45 ± 2.7; range between P14 and P114) were used. The sex of animals is not reported.
Mice were anaesthetized with isoflurane and then transcardially perfused with ice-cold, gassed artificial cerebrospinal fluid (aCSF) containing (in mM) 127 NaCl, 25 NaHCO3, 10 D-glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, and 1 MgCl2. The brain was then resected and 300 µm-thick slices were obtained using a vibratome (Leica VT1200S) in an ice-cold, gassed sucrose-cutting solution containing (in mM): 210 sucrose, 25 NaHCO3, 10 D-glucose, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4 and 1 sodium pyruvate. The slices were then incubated in gassed aCSF at 35 °C for 30 min and subsequently kept at room temperature for up to 6 hours.
Coronal slices were made for the following brain the dorsolateral striatum, barrel cortex (BC), basolateral amygdala (BLA), globus pallidus externa (GPe), medial prefrontal cortex (mPFC), suprachiasmatic nucleus (SCN), substantia nigra pars compacta (SNc), ventral thalamus (Th) and primary visual cortex (V1). Slices containing CA1 region of the hippocampus (CA1) were cut horizontally; those containing the cerebellum (Cb) were cut sagittally.
Whole-cell voltage-clamped recordings were performed using a MultiClamp 700B amplifier (Molecular Devices) controlled with custom software written in MATLAB. Electrophysiological signals were filtered at 2 kHz before being digitized at 20 kHz. Slices were perfused at room temperature with gassed aCSF containing CPP (10 µM). Recording pipettes (3–5 MΩ) were pulled from borosilicate glass (G150F-3; Warner Instruments) using a model P-1000 puller (Sutter Instruments). Series resistance was 10–25 MΩ. The internal solution contained (in mM): 126 Cs-gluconate, 10 HEPES, 5 Na-phosphocreatine, 0.5 Na-GTP, 4 Na-ATP, 5 TEA-Cl, 5 EGTA and 4 QX-314 bromide with an osmolarity of 280-290 mOsmol/kg and pH ~7.2 adjusted with CsOH. The junction potential was calculated to be -14 mV, as calculated using JCal from Clampex software (Molecular Devices). Voltages were not corrected for the theoretical liquid junction potentials. For IV curves recordings, we decreased the chloride concentration in the internal solution (in mM): 126 Cs-gluconate, 10 HEPES, 8 Na-phosphocreatine, 0.3 Na-GTP, 4 Mg-ATP, 1 EGTA and 1 QX-314 chloride with an osmolarity of 280-290 mOsmol/kg and pH ~7.3 adjusted with CsOH.
For miniature postsynaptic currents recordings, TTX (1 μM) and CPP (10 µM) was added to the bath to block action potentials and NMDA currents, respectively. 20-minute traces were recorded in voltage-clamp mode and mEPSCs and mIPSCs events were detected using a built-in template matching feature in Clampfit (verions 10.7; Molecular devices). Recordings were done in acute slices from C57BL/6NCrl mice. We also performed miniature recordings in SPNs from the dorsolateral striatum from D1-Tdtomato mice. dSPNs were identified by the presence of fluorescence, whereas iSPNs were non-fluorescent neurons that were not ChINs, which can be identified via their uniquely large soma.
Biphasic minis were visually identified via their unique shape. We only included biphasic minis in which the mEPSC peak was connected to the mIPSC peak by a smooth continuous line as has been observed elsewhere^16,17^. In other words, we excluded biphasic events in which the decay of the mEPSC to the rise of the mIPSC (for straight biphasic minis) or the decay of the mIPSC to the rise of the mEPSC (for reverse biphasic minis) was discontinuous. For Supplementary Fig. 2, biphasic minis were detected using the built-in template matching feature in Clampfit (Molecular Devices). Templates were derived from filtered (low pass gaussian filter with −3 dB cutoff at 1 kHz) representative single biphasic minis (both straight and reverse) identified by visual inspection. The selection was based on comparable downward and upward amplitudes and the shapes representing the average of visually detected events. In pharmacological experiments, 10 μM gabazine and 10 μM NBQX were added to the aCSF to block GABAAR and AMPAR, respectively. In neuromodulation experiments, adenosine (20 µM) or serotonin (10 µM) was added to the perfusion solution.
For recording in different brain regions and cell types, identification of the neurons was based mostly on their characteristic morphology and location. For each region, cells were held at a holding potential that maximized the detection of biphasic minis (VH in mV): BC, -25; BLA -30; CA1 -25; Cb -35; ChIN -25; GPe -40; mPFC -30; SCN -30; SNc -30; Th -30; and V1 -30. For recordings in Purkinje cells, 100 µM CdCl2 was added to the bath to avoid calcium spikes and calcium oscillations.
6-hydroxydopamine (Tocris) injections were performed in WT mice at 8–9 weeks of age. Animals were anesthetized using 2% isoflurane, mixed with oxygen, and placed in a stereotaxic frame. After making a small incision to expose the scalp, the skull was cleaned above bregma and lambda and a dental drill was used to make a small craniotomy above the SNc. 6-OHDA was dissolved in saline (0.9% w/v NaCl with 0.02% w/v ascorbic acid), to a final concentration of 5 μg/μL, immediately before use to minimize the oxidative effects on 6-OHDA. Injections were performed in 3 sites per animal (600 nL/site), at a rate ~35 nL/min into the SNc (at AP 2.4 mm and ML 1.25 mm from bregma, and DV 3.8, 4.0, and 4.2 mm, respectively). Injections were performed using a micropipette pulled using a micropipette puller (P-97; Sutter Instruments). Desipramine (25 mg/kg delivered IP) was given 30 min prior to 6-OHDA infusion to block uptake of the toxin by noradrenergic neurons.
Four days after unilateral 6-OHDA injection, electrophysiological recordings were performed in both hemispheres. Dopaminergic lesions were confirmed by immunohistochemistry of striatal tyrosine hydroxylase (TH) which showed complete depletion of TH in the striatal hemisphere ipsilateral to the 6-OHDA lesion.
Four days after unilateral 6-OHDA injection, the mouse was anaesthetized with isoflurane and fixed via cardiac perfusion with 4% paraformaldehyde. Brain was dissected out and post-fixed in 4% paraformaldehyde overnight. Coronal sections (50 μm) were sliced to free-floating sections using a vibratome (Leica VT1200S). Sections were incubated in blocking solution for 30 min at room temperature. Primary antibody (Anti-tyrosine hydroxylase antibody, from rabbit) was diluted 500× and slices were incubated on a shaker overnight at 4 °C. Slices were rinsed three times in PBS. Secondary antibody (Anti-rabbit Alexa fluor 488, from goat) was diluted 500× and slices were incubated on a shaker at room temperature covered by foil for 2 h. Slices were rinsed again three times in PBS. Sections were mounted on to slides and coverslip with fluoromount (Sigma #F4680) and edges were painted with nail polish.
The biphasic minis were fit using averaged mEPSC and mIPSC traces (those in Supplementary Fig. 1A, −30 mV) with freedom in amplitudes and the relative onset delays. The fitting was carried out in MATLAB using custom-made algorithms based on nonlinear least-squares data fitting by the Gauss–Newton method with the Levenberg-Marquardt adjustment. Nearly all traces (181 out of 184 straight biphasic minis and all 151 reverse biphasic minis) were well fit by this approach. The averaged onset delay was ~10 ms for both types of biphasic minis. We therefore used this value as the time window to calculate chance probability (below).
To determine whether the observed number of biphasic minis events was due to chance, we calculated the expected probability of two independent events (one mEPSC and one mIPSC) occurring in the same time window (10 ms). The independent probability (i.e. chance) was calculated as the joint probability of finding a mEPSC and a mIPSC in a 10 ms time
Where PE corresponds to the chance of a mEPSCs found in the window and PI corresponds to the chance of mIPSCs found in the same window. The real chance of finding a biphasic mini in the same window was normalized to that of the independent probability.
Recordings were made from coronal slices (300 µm thickness) containing the dorsolateral striatum from DAT-Cre/Ai32 double-het mice, which expresses ChR2(H134R)-EYFP in dopaminergic axons. Minimal optogenetic stimulation of dopaminergic axons was evoked by applying a single short pulse (1 ms) of blue light (LED) under widefield illumination using an 60x objective at an empirically determined low intensity light. Light was applied to a field (440 μm) centered on the recorded neuron, and the intensity was adjusted so that failure and apparent quantal synaptic responses could be observed in different trials. In voltage-clamp mode, cells were held at a holding potential (VH) of -45 mV to record biphasic responses (EPSCs and IPSCs). At this VH, mEPSCs and mIPSCs and upward and downward responses of biphasic currents had similar amplitude. Pulses were applied every 30 s and 30 trials were recorded.
For minimal stimulation experiments, the expected percentage of biphasic PSCs generated by chance from independent EPSCs and IPSCs were calculated from the following
All imaging experiments were carried out on live samples. Neurons were imaged using a custom-built two-photon microscope under 60× 1.0 NA water-immersion objective (up-right) controlled by ScanImage software (Vidrio). Acute brain slices were imaged in a chamber perfused with gassed ACSF. At the end of the electrophysiological recordings, neurons were filled with Alexa Fluor 594 (50 µM) by passive diffusion from the recording pipette. Samples were excited using a MAITAI HP Ti:Sapphire laser (Newport) at 960 nm, red fluorescence was isolated using a dichroic (Chroma 565DCXR) and band-pass filters (Semrock FF01-630/92). Images were acquired at 512 × 512 pixel density and a z-step size of 1 µm. Image analyzes were performed using custom software written in MATLAB.
All experiments were conducted multiple times (typically ≥3) in different animals or cells. Quantification and statistical tests were performed using custom software written in MATLAB. Paired and unpaired t-tests were used for paired and unpaired data, respectively, unless otherwise noted. For the analysis of probabilities from non-normally distributed data, a logarithmic transformation was applied, followed by a paired t-test. Poisson distribution was calculated for λ = 2. Some data was removed due to low sample size (< 3 events per cell). Averaged data are presented as mean ± SEM, unless noted otherwise. Throughout the paper, “n” indicates the number of neurons. In all figures, *p ≤ 0.05 and is statistically significant, **p ≤ 0.01, and ***p ≤ 0.001.
Reagent list.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
We thank Drs. Paul Brehm, Gary Westbrook, John Williams, Tianyi Mao, Michael Muniak, and James Jones for critical comments on the manuscript. We thank all members of the Mao and Zhong laboratories at the Vollum Institute for constructive discussions. This work was supported by three NIH BRAIN Initiative awards (RF1MH130784, RF1NS133599, and R01NS104944) and an NINDS R01 (R01NS127013) to H.Z.
C.C. made the initial observation of biphasic minis. C.C. and H.Z. designed the experiments. C.C performed the experiments and data analyzes. L.M performed 6-OHDA injections. M.Q. maintained mouse husbandry. H.Z. and C.C wrote the manuscript. All authors edited and commented on the manuscript. H.Z. secured the funding and supervised the project.
Communications Biology thanks Steve Shabel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Christian Wozny and Rosie Bunton-Stasyshyn. A peer review file is available.
Source data will be provided with this paper (Supplementary Data 1–12). Original raw data will be provided upon request.
This study did not develop broadly applicable code or algorithm. Custom MATLAB scripts for specific data analysis will be made available upon request.
The authors declare no competing interests.
The online version contains supplementary material available at 10.1038/s42003-024-07198-y.
Source data will be provided with this paper (Supplementary Data 1–12). Original raw data will be provided upon request.
This study did not develop broadly applicable code or algorithm. Custom MATLAB scripts for specific data analysis will be made available upon request.