Intrinsic membrane properties of striatal neuronal subtypes
Whole-cell recordings were made of MSNs from the striatum of NT (n
=85), hWT (n
=81) and hMT (n
=92) mice, identified by means of electrophysiological criteria (Kita et al., 1984
). MSNs from either NT or hWT and hMT mice did not exhibit firing activity at rest and, upon depolarizing current pulses showed membrane rectification and tonic action potential discharge (). Resting membrane potential (RMP), action potential amplitude, input resistance were similar in the three strains (; ; p
>0.05) and did not differ from those previously described for mouse MSN (Kitada et al., 2007
; Centonze et al. 2003
). The current–voltage relationship did not show significant differences among groups (; p
>0.05). shows a recorded MSN, double-labelled for biocytin and DARPP-32, showing the peculiar morphological features of a spiny neuron.
Fig.1 Characterization of medium spiny neurons (MSNs) and fast-spiking (FS) interneurons. (A) Superimposed sample recordings in current-clamp mode showing voltage responses to both depolarizing and hyperpolarizing current steps (300 pA, 600 ms) inindividual (more ...)
Membrane properties of medium spiny neurons (MSNs) and of Fast-Spiking (FS) GABAergic interneurons recorded from striatal slices of non-transgenic (NT), wild-type (hWT) and mutant (hMT) mice
Intrinsic membrane properties were used to identify striatal GABAergic FS interneurons and to distinguish them from other striatal neuronal subtypes (Tepper et al., 2004
) (; ). Compared to MSNs, FS interneurons (n
=29) had a more depolarized RMP and higher input resistance. FS interneurons were silent at rest, and upon brief depolarization exhibited early, brief spikes (width at half amplitude<1 ms), and rapidly peaking after hyperpolarizations (, ). In response to depolarizing pulses of longer duration, FS interneurons showed either a brief burst of spikes, or an early spike followed by a tonic firing discharge without accomodation (; ). The membrane properties of FS interneurons did not differ from those measured in hWT (n
=18) and hMT mice (n
Spontaneous glutamate- and GABA-dependent synaptic events
The activity of striatal MSNs is driven both by cortical and thalamic glutamatergic inputs. Thus, we measured sEPSCs, which are considered a reliable indicator of glutamatergic activity, from MSNs of NT and transgenic mice. MSNs were clamped at -80 mV and sEPSCs were recorded in the presence of the GABAA
receptor antagonist bicuculline (10 μM). A combination of NMDA and AMPA glutamate receptor antagonists, MK-801 (30 μM) and CNQX (10 μM) fully blocked spontaneous synaptic events (not shown). Most of the sEPSCs recorded from NT MSNs (n
=11) had amplitude and frequency ranging between 5 and 40 pA, and 0.6 and 7 Hz, respectively and did not differ from hWT (n
=10) and hMT (n
=11) MSNs (Fig. S1
>0.05). Kinetic properties, such as rise time, decay time constant and half width, were similar in the three groups (not shown; p
As an indicator of striatal GABAergic synaptic activity, in a parallel set of experiments we measured inhibitory GABAergic synaptic currents (sIPSCs). Recordings with CsCl-containing pipettes at -75 mV as HP, and in the presence of MK-801 (30 μM) and CNQX (10 μM), allowed detection of bicuculline-sensitive sIPSCs (10 μM). The amplitude (7–40 pA) of sIPSCs recorded from NT MSNs (17±2.2; n=15) was similar to that measured in hWT (20.8±3.2; n=10) and hMT (21±3; n=11) mice (; p>0.05). The kinetic characteristics of sIPSCs were indistinguishable among the three groups (; p>0.05). Conversely, the mean frequency of sIPSCs was significantly increased in hMT mice (3.01±0.5 Hz), compared to both hWT (2.2± 0.2 Hz) and NT (; 1.9±0.2; p<0.05), suggestive of an increased GABAergic input onto MSNs.
Fig. 2 Changes in GABA-dependent spontaneous inhibitory synaptic currents (sIPSCs). (A) Voltage-clamp recordings from MSNs showing sIPSC (downward deflections) in the presence of MK-801 (30 μM) and CNQX (10 μM) in the bathing solution, in slices (more ...)
To analyze further GABA-dependent synaptic activity, we recorded miniature IPSCs (mIPSCs), in the presence of TTX, to block actionpotential dependent GABA release. As expected, TTX homogeneously reduced mean frequencies but not amplitudes of synaptic events, if compared to sIPSCs (~72% of control). However, we found a significantly higher frequency, but not amplitude, of mIPSCs recorded from MSNs of hMT (1.7±0.3 Hz; n=11), compared to hWT (1±0.14 Hz; n=10) and NT mice (0.99±0.1 Hz; n=12) (; p<0.05), suggesting that the increase in GABA synaptic events relies, at least partly, upon action potential-independent GABA release.
Fig. 3 Properties of miniature inhibitory synaptic currents (mIPSCs). (A) Sample recordings of mIPSCs recorded in TTX (1 μM), MK-801 (30 μM) and CNQX (10 μM), in non-transgenic (NT) MSNs (a), hWT(b) and hMT (c) mice. Holding potential: (more ...)
Spontaneous synaptic events in fast-spiking interneurons
Striatal FS interneurons constitute an interconnected network of GABAergic cells that exerta powerful feed-forward inhibitory control on MSNs (Koós and Tepper, 1999
; Tepper et al., 2004
). The FS interneurons are, in turn, driven primarily by excitatory afferents from neocortex. To explore the possibility that altered drive to FS interneurons was responsible for the increase in synaptic GABAergic events recorded from MSNs, we measured sEPSCs from FS interneurons from NT and transgenic mice. Measurement of mean frequency and amplitude of glutamatergic sEPSCs did not reveal significant changes either in hWT (8±2.2 Hz; 8.2±0.9 pA; n
=6), in hMT (8.8±2.4 Hz; 7.1±0.8 pA; n
=8) mice, or compared to NT animals (; 9.4±3 Hz; 8±0.8 pA; n
>0.05). Likewise, no significant difference was measured among sIPSCs (NT=4.2±0.8 Hz, 15.5±1.5 pA, n
=7; hWT=4.3±0.9 Hz; 14.4±2 pA, n
=6; hMT=4.2±1.1 Hz; 15.5±2 pA, n
Fig. 4 GABAergic and glutamatergic spontaneous synaptic events from FS interneurons. (A) Representative recordings of sIPSCs from FS interneurons from NT (a), hWT (b) and hMT (c) mice. Cells were clamped at -70 mV, in the presence of MK-801 (30 μM) and (more ...)
Likewise, analysis of kinetic properties for both synaptic events did not differ among the three groups of mice (data not shown, p>0.05). Together, these results do not support a major involvement of altered excitatory drive to FS interneurons in the elevation of GABAergic synaptic activity observed in hMT mice.
Effects of D2 DA receptor activation on sIPSCs
In normal conditions, striatal GABA synthesis and release is under the inhibitory control of D2 DA receptors expressed on GABAergic nerve terminals (Delgado et al., 2000
; Guzmán et al., 2003
; Centonze et al., 2003
). To address whetherD2 receptor dysfunction was responsible for the increase in sIPSC frequency, we measured the response of MSNs and FS interneurons to the D2-like receptor agonist quinpirole. Although quinpirole can activate both D2 and D3 receptor proteins, the dorsal striatum lacks D3 receptors, and therefore the effects of quinpirole in this region on synaptic currents are presumably mediated by D2 receptors (Le Moine and Bloch, 1996
; Guzmán et al., 2003
In slices from either NT (n
=19) or hWT mice (n
=17), bath application of quinpirole (1–30 μM, 10 min) caused no consistent changes in membrane properties of the recorded cells, and dose dependently reduced the frequency, but not the amplitude of sIPSCs recorded from MSNs (; p
<0.05). After 10 μM quinpirole, sIPSC frequency was 83.2±5% of control (pre-drug) in NT mice, 76.3±4.2% in hWT mice, whereas amplitude was unaffected (; NT=97.1 ± 8%; hWT=86.3±12%, p
>0.05). The quinpirole effect on sIPSC frequency was prevented by pretreatment with the D2 receptor antagonist l
-sulpiride (3 μM, 98.7±1.1%, and 101.5±2.2% in NT and hWT, respectively; n
=5 for both groups; data not shown). On the contrary, 10 μM quinpirole failed to modify either the frequency (102.6±5.3%) or the amplitude (99.2±6.2%) of GABA-dependent sIPSCs recorded from hMT MSNs, as well as at all concentrations tested (; n
>0.05). Moreover, quinpirole (10 μM; 10 min) affected neither frequency or amplitude of mIPSCs in NT (n
=8) as well as in transgenic mice (Fig. S2
=5 and 7 for both hWT and hMT, respectively; p
>0.05), a result which is consistent with the notion that mIPSCs are calcium-independent events (Zhu and Lovinger, 2005
; Centonze et al., 2008
), whereas the D2-dependent inhibition involves calcium channels (Yan et al., 1997
). We also tested the possibility of an inhibitory tone mediated by D2 receptors on GABA transmission in NT and hWT mice by bathing the slices with l
-sulpiride. Both frequency and amplitude of sIPSCs were not affected by l
-sulpiride (3 μM, 10 min) (NT=1.7±0.6 Hz; 18.1±3 pA; hWT=2±0.4 Hz; 19.7±3.3 pA; n
=10 for each genotype, p
>0.05; data not shown).
Fig. 5 D2 dopamine receptor activation fails to inhibit sIPSCs in hMT mice. (A) Sample sIPSCs recorded before and 10 min after quinpirole application (10 μM) in MSNs recorded from NT animals (a,a1), as well as from hWT (b,b1) and hMT mice (c,c1). Cells (more ...)
To ascertain if the loss of modulation was specific to D2 receptors or rather it reflects a more generalized alteration of other Gi/Go coupled receptor at GABAergic synapses, we examined the effect of both the GABAb receptor agonist baclofen and the CB1 receptor agonist HU 210 on sIPSCs. HU 210 (1 μM, 10 min) reduced sIPSC frequency homogeneously in the three strains (; NT=54.3±3.1%; hWT=52.1±3.6%; hMT=57.4±3.6%; n=5 for each genotype, p<0.05). Conversely, sIPSC amplitude was unaffected (; NT=102.1±15.2%; hWT=111±13.1%; hMT=105.2±17%; n=5 per genotype; p>0.05). Similarly, 10 μM baclofen significantly decreased sIPSC frequency (; NT=74±5.1%; hWT=75±6.3%; hMT=69.2±3.4%; n=5 per genotype; p<0.05), but not the amplitude (; NT=97.1±8%; hWT=88.3±11.2%; hMT=99±6.4%; n=5 per genotype; p>0.05), confirming the specificity of D2 receptor alteration. In addition, to unmask a possible tonic endocannabinoid release, slices were bathed in the presence of a CB1 receptor antagonist, AM251 (10 μM, 10–15 min). Both frequency and amplitude were unaffected by AM251 in the three strains of mice (frequency:NT:82±10%; hWT:84.4±16.8%; hMT: 81.2±14.4%; p>0.05; for amplitude, NT: 94.8±9.8%; hWT: 94.5±8.9; hMT: 92.3±7.9%; n=4 per genotype, data not shown).
Fig. 6 CB1 and GABAb receptors preserve their inhibitory action in hMT mice. Representative voltage-clamp recordings showing the ability of the CB1 receptor agonist HU 210 (1 μM) to suppress sIPSCs from MSNs of NT (a), hWT (b) and hMT (c) mice. (B) The (more ...)
Finally, we analyzed the effect of D2 activation on FS interneurons. No significant change was observed on holding current and firing rate of the recorded interneurons after bath-application of quinpirole. We were unable to detect a modulatory effect of quinpirole (1–10 μM) on sIPSCs of FS interneurons in slices from NT, hWT or hMT mice (; n=7, 6, and 6, respectively; p>0.05). After bath application of 10 μM quinpirole, no significant change was measured (Frequency: 102.4±3.5% for NT; 100.8±5.2% for hWT; 99.5±3.6% for hMT; amplitude: 101.1±5.1% for NT; 102.6±4% for hWT; 93.6±5.9% for hMT; p>0.05).
Fig. 7 FS interneurons are insensitive to the quinpirole-mediated inhibition. A. Sample traces showing the inability of quinpirole (10 μM) to inhibit sIPSCs recorded from FS interneurons of hMT mice. B. The plots summarize the lack of effect of quinpirole (more ...)
Absence of D2 DA-dependent inhibition on evoked IPSCs from MSNs and FS interneurons
In another group of experiments, eIPSCs were elicited by intrastriatal electrical stimulation. These synaptic currents were recorded with CsCl-filled pipettes, with cells clamped at 0 mV.
Mean amplitude values, in response to synaptic stimuli able to elicit 30–50% of the maximal eIPSC amplitude (see methods), were 65±15 pA (NT; n=8), 59.5±16.6 pA (hWT; n=9) and 61±17 pA (hMT; n=10).
Bath application of quinpirole (1–30 μM,10 min) did not cause any change in RMP or input resistance of the MSNs, but reduced the amplitude of the eIPSC recorded both from NT and hWT mice (; 67.7±2.1%, 66.1±2.3% of control, respectively at 10 μM; n=12 for each genotype; p<0.05). This effect was fully prevented by pretreatement with l-sulpiride (3 μM, not shown). In contrast, in MSNs from hMT mice quinpirole failed to modify the eIPSC amplitude (; 98±4.6%; n=12; p>0.05).
Finally, we examined the effect of quinpirole also on eIPSCs in FS interneurons. Evoked IPSCs were recorded in the same experimental conditions as described for MSNs. The mean amplitudes measured in FS neurons were 47.2±11.5 pA (n=14) for hMT mice, 49.1±12 pA, (n=12) for hWT, and 61.5±13 pA in NT mice (n=13) (). In FS neurons from NT and hWT animals, perfusion of the slice with quinpirole dose-dependently reduced the eIPSC amplitude (; at 10 μM quinpirole: 68.2±3.2%; 68.3±3%, respectively; p<0.05), without modifying membrane properties. Conversely, quinpirole failed to alter either the amplitude or kinetic properties of eIPSCs in FS interneurons from hMT mice (; 10 μM quinpirole: 86.9 ±1.6%; p>0.05).