3A). (2011) using channel rhodopsin activation in striatal MSNs suggests MSN‐MSN collaterals occur at a rate of 63%. 72 0 R Cells were visualized using a Nikon TE‐2000S inverted microscope with a 40× phase contrast objective (Tokyo, Japan) and tdTomato and EGFP‐expressing neurons were selected. Interestingly, D2 MSNs exhibited larger currents in response to THIP compared with D1 MSNs, suggesting a greater expression of extrasynaptic GABAA receptors in D2 MSNs basally in vitro (Fig.
The absence of natural targets for MSNs in culture may explain the high rate of autapse formation, as is exemplified with in vitro microislands (Bekkers, 2005). Crossing these two strains resulted in healthy mice pups, a subset of which coexpressed tdTomato in D1 cells and EGFP in D2 cells. Previous studies have shown that DA regulates the efficacy of MSN collaterals (Taverna et al., 2008). 5A,B).
It remains to be determined whether the increase in synaptic sites in treated D2 MSNs is due to a morphological change in dendritic branching (Day et al., 2006), or whether it is due to a facilitation of GABAA receptor insertion at newly formed inhibitory synapses. endobj DOI: https://doi.org/10.1523/JNEUROSCI.0692-13.2013. D1 receptor containing MSNs (D1 MSNs) project directly to the substantia nigra pars reticulata (SNpr), and D2 receptor containing MSNs (D2 MSNs) project indirectly to the SNpr via the external segment of the globus pallidus (Gong et al. Anatomical reconstruction was achieved by including 1% biocytin in the intracellular solution of a subset of neurons (Partridge et al., 2009). A correlated increase in the frequency of spontaneous (Fig. Although mIPSCs may be derived from presynaptic terminals from cortical GABAergic neurons, striatal interneurons or MSNs, the occurrence of mIPSCs indicates the successful innervation and presence of postsynaptic GABAA receptors. D2R activation increases number of presynaptic terminals. At postnatal day 0–1 (p0‐1), male and female pups were decapitated in accordance with the guidelines of the American Veterinary Medical Association Panel on Euthanasia and the Georgetown University Animal Care and Use Committee. B, Summary plots of gephyrin puncta density (left) and puncta intensity (right) of identified GFP+ and GFP− dendrites (**p < 0.01; Mann–Whitney U test). These benefits come with inherent limitations: dissociated culture models disrupt the natural development of cytoarchitecture, potentially altering cellular and network phenotypes. %���� MSNs consistently displayed rapid and homogenous latency to peak (4.3 ± 0.2 ms), and low rates of failures (8.9 ± 2%), suggesting that eIPSCs were a result of direct monosynaptic connections (n = 50 cells from six culture preparations). Together, our data indicate that chronic D2R activation results in a greater synaptic efficacy via a coordinated increase of synaptic GABAA receptor clusters and GABA release sites. Yellow arrows mark the stimulation pulse. 0000001629 00000 n The coverslips had been previously incubated in Basal Eagle's medium (Invitrogen). startxref The decay phase of the averaged eIPSC was fit to a double exponential equation. Coverslips treated in parallel with 500 nm quinpirole exhibited 75% coupling in 28 pairs (p = 0.156 when compared with nontreated D2 MSN pairs; χ2 test).
B: Two juxtaposed granule neurons each send a single unramiﬁed axon into the longitudinal hippocampal axis. As D1 and D2 receptors are expressed in the cortex by P1 (Fig. However, a recent study by Chuhma et al. The characteristics of MSNs in the presence and absence of treatments were also confirmed in cultures harvested from two separate mouse strains: drd2-cre;rosa26-tdTomato (Madisen et al., 2010) and npy-EGFP;drd1a-tdTomato (Shuen et al., 2008; Partridge et al., 2009). Brains were harvested for cultures at P0‐1. 3B), as well as a decrease in coefficient of variation for postsynaptic D2 MSNs (0.50 ± 0.05, n = 45 cells to 0.34 ± 0.02, n = 55 cells, p = 0.007; Mann–Whitney test) but not D1 MSNs (0.42 ± 0.04%, n = 30 cells to 0.39 ± 0.02, n = 27 cells, p = 0.867; Mann–Whitney test). Thus, together, the increase in mIPSC frequency and GABA sensitivity point to an increase in number of postsynaptic GABAA receptors with treatment. 0000022229 00000 n Using paired whole‐cell patch‐clamp recordings from identified MSNs at 14 days in vitro, we are able to detect a much higher rate of inhibitory functional synapses than what has been previously reported in slice recordings. B, Summary plots compare charge transfer, fitted decay time constant (τw) and rate of failure, separated by experimental condition (n = 13 and 15 for D1–D1 nontreated and treated MSNs, respectively; n = 19 and 16 for D2–D1 nontreated and treated MSNs, respectively; n = 16 and 21 for D1–D2 nontreated and treated MSNs, respectively; n = 22 and 33 for D2–D2 nontreated and treated MSNs, respectively).
These experiments suggest that the increased sensitivity to GABA observed in quinpirole-treated D2 MSNs is not related to changes in β2/3 or δ subunit expression. The characteristics of MSNs observed in cultures from drd1a-tdTomato; drd2-EGFP remained consistent across three additional mouse strains and genotypes (drd2-EGFP, drd2-cre;rosa26-tdTomato, and npy-EGFP;drd1a-tdTomato; data not shown). Borosilicate glass capillaries (Wiretrol II, Drummond, Broomall, PA) were pulled on a two‐step vertical pipette puller (PP‐83; Narishige, Tokyo, Japan). <> << We first examined whether chronic D2R activation with 10 μm quinpirole altered the rate of functional synapse formation between MSN pairs. When [Ca2+]o was increased to 2 mm [Ca2+]o, nontreated D2 MSNs again displayed an increase in eIPSC amplitude variance and amplitude mean (Fig. 6A). After 15 min in a 37°C water bath shaking at 180 rpm, an equal volume of buffer solution containing 0.001% DNAase, 0.01% trypsin inhibitor, and 0.01% MgSO4 was added to the suspensions and centrifuged at 2000 rpm for 5 min. Miniature IPSCs (mIPSCs) were isolated with the presence of TTX and NBQX, and events were detected using semiautomated threshold‐based software (Mini Analysis, Synaptosoft, Fort Lee, NJ). 0000010008 00000 n A nerve cell is called the neuron. Scale bars: x-axis, 150 ms; y-axis, 100 pA. As the effects of the treatment on eIPSCs were observed only when the postsynaptic target was a D2 MSN, we hypothesize that D2R activation preferentially affects postsynaptic D2 MSNs, independent of the presynaptic partner. /ExtGState<> Similarly, at 3 μm GABA, currents were 94 ± 33 pA, n = 12, and 250 ± 65 pA, n = 11, in nontreated and treated D2 MSNs, respectively (p = 0.032; Mann–Whitney test). Puncta density and fluorescence intensity of immunoreactive puncta were calculated by thresholding images at two times background. 4) and the increased sensitivity of treated D2 MSNs to low doses of GABA that parallels increases in gephyrin clusters (Fig. Mg (5), GTP.Na (0.2), and ethylene glycol tetraacetic acid (0.5), adjusted to pH 7.2 with KOH (IS1). Tel: 202‐687‐1567 Fax: 202‐687‐6441 E‐mail: email@example.com. endobj 2003; Shuen et al. Flumazenil, a potentiator of α4, β3, and γ2 subunit-containing GABAA receptors (Ramerstorfer et al., 2010), failed to prolong IPSC decay in either condition, excluding a role for the α4 subunit (data not shown). The use of transgenic mice that allow for live identification of two populations of neurons provides us an additional, straightforward internal control. The ideas and opinions expressed in JNeurosci do not necessarily reflect those of SfN or the JNeurosci Editorial Board.
We utilize bacterial artificial chromosome (BAC) transgenic mice that express td‐Tomato downstream of the D1 receptor promoter and enhanced green fluorescent protein (EGFP) downstream of the D2 receptor promoter to concurrently distinguish between the two MSN subtypes (Gong et al. 3E and H) (Randall et al. Recordings of cells which had leak currents of greater than 100 pA or RMP of less than −50 mV were discarded. These high‐frequency MSN collaterals have the ability to shape the firing patterns of neurons they are coupled with and are therefore important in striatal circuit output. 0000021538 00000 n
We observed that diazepam significantly prolonged mIPSC decay: in nontreated D2 MSNs, τw increased from 25 ± 2 to 33 ± 1 ms, n = 12 (p < 0.0001; paired t test); in treated D2 MSNs, τw increased from 27 ± 3 to 39 ± 3 ms, n = 8 (p < 0.0001; paired t test). Improper striatal function results in a slew of pathological states including Parkinson's disease, and striatal collaterals have been largely implicated with changes in dopamine levels (Taverna et al. Meninges were removed and brains were checked for fluorescence using Dual Fluorescent Protein Flashlight (NIGHTSEA, Bedford, MA). EGFP expression was used to guide the exact dissection of striata from brains expressing both fluorophores. Three to five neurons were analyzed per condition in three separate cultures and results were plotted as the number of intersections per 10 μm concentric circle from the soma. Neurons were pharmacologically stimulated between DIV 18 and 21. We then chronically treated the cells with 10 μm sulpiride plus 10 μm quinpirole for 72 h. A smaller subset of coverslips was treated with a low dose of quinpirole (500 nm). w�|���e^����bcnF�&5C�U����t���B�2%Ef�?C����o@��ⷲ��x�N��,�u�"S�֤��v(��X�S�����OQ��k�U��o �bYZ���:�p�� Puncta measures were quantified with a custom macro for NIH ImageJ software (courtesy of F. Vanevski). Slices containing both striatum and substantia nigra were mounted on glass coverslips with VectaShield and visualized with and upright E600FN Nikon microscope. Second, quinpirole-treated D2 MSNs display increased dendritic complexity, a morphological change that may enhance the likelihood of synapse formation.
1998), the water soluble and quickly reversible nature of BMR application outweighed the potential non‐GABAergic effect.
Ultimately, it is important to keep in mind the limitations of each preparation when interpreting results. Scale bar, 30 μm. 0000018961 00000 n In addition to synaptic currents, we discovered the frequent occurrence of what appeared to be autaptic currents immediately following stimulation (Fig. Publication of an advertisement or other product mention in JNeurosci should not be construed as an endorsement of the manufacturer’s claims. /O 49 6B). 4D) and reports a reduction in soma diameter (Fig. Quinpirole, an agonist for D2/3 receptors, was used to chronically activate D2Rs. Charge transfer and time to decay (τw) increased in treated D2 MSNs, regardless of presynaptic partner (Fig. The authors declare no competing financial interests. Figure 6 is an example of two MSNs that are unidirectionally coupled and in current clamp configuration. As we did not detect any significant differences between strains, these data were excluded from the results section for consistency. Each axon terminates Although we cannot exclude a preferential increase in other GABAA receptor subtypes, or postsynaptic modifications of existing receptors, the most plausible explanation is an increase in the density of GABAA receptor clusters with quinpirole treatment. However, the slower decay of eIPSCs compared with mIPSCs suggests they reflect asynchronous summation of multiple synaptic currents produced at distinct sites (Mody et al., 1994). The equilibrium potential (ECl−) for Cl− in this condition was −71.4 mV.
<> In support of this observed increase in connectivity, the likelihood in which two D2 neurons were reciprocally coupled was also significantly increased: while reciprocal connections were found in 26 of 67 (39%) cells analyzed in nontreated conditions, reciprocal connections increased to 60 of 97 (62%) analyzed cells after chronic D2R activation (p = 0.004; χ2 test). ce`a�X���T��C&�1���FNG!���� ��0��!�Ġٷ����ǹ��������_��O��u���;R������~���'���~?����쇑�2����J>�x����_�H0P �8H�2� �@� Vo���b ��Kb�O����b��8�4���A�F���"�F����n� All recordings were done at room temperature (RT) in extracellular solution (ES) continuously perfused at a rate of 2.5 ml/min. 1990), reducing or removing serum (Ahmed et al.
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