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, 30 (20), 6999-7016

Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum

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Electrophysiological and Morphological Characteristics and Synaptic Connectivity of Tyrosine Hydroxylase-Expressing Neurons in Adult Mouse Striatum

Osvaldo Ibáñez-Sandoval et al. J Neurosci.

Abstract

Whole-cell recordings were obtained from tyrosine hydroxylase-expressing (TH(+)) neurons in striatal slices from bacterial artificial chromosome transgenic mice that synthesize enhanced green fluorescent protein (EGFP) selectively in neurons expressing TH transcriptional regulatory sequences. Stereological cell counting indicated that there were approximately 2700 EGFP-TH(+) neurons/striatum. Whole-cell recordings in striatal slices demonstrated that EGFP-TH(+) neurons comprise four electrophysiologically distinct neuron types whose electrophysiological properties have not been reported previously in striatum. EGFP-TH(+) neurons were identified in retrograde tracing studies as interneurons. Recordings from synaptically connected pairs of EGFP-TH(+) interneurons and spiny neurons showed that the interneurons elicited GABAergic IPSPs/IPSCs in spiny neurons powerful enough to significantly delay evoked spiking. EGFP-TH(+) interneurons responded to local or cortical stimulation with glutamatergic EPSPs. Local stimulation also elicited GABA(A) IPSPs, at least some of which arose from identified spiny neurons. Single-cell reverse transcription-PCR showed expression of VMAT1 in EGFP-TH(+) interneurons, consistent with previous suggestions that these interneurons may be dopaminergic as well as GABAergic. All four classes of interneurons were medium sized with modestly branching, varicose dendrites, and dense, highly varicose axon collateral fields. These data show for the first time that there exists in the normal rodent striatum a substantial population of TH(+)/GABAergic interneurons comprising four electrophysiologically distinct subtypes whose electrophysiological properties differ significantly from those of previously described striatal GABAergic interneurons. These interneurons are likely to play an important role in striatal function through fast GABAergic synaptic transmission in addition to, and independent of, their potential role in compensation for dopamine loss in experimental or idiopathic Parkinson's disease.

Figures

Figure 1.
Figure 1.
Electrophysiological properties of neostriatal and substantia nigra pars compacta neurons from BAC transgenic EGFP–TH+ mice. A, Typical striatal SPN. Note the extremely low input resistance, strong inward rectification, lack of spontaneous activity, and depolarizing ramp in response to depolarizing current pulse leading to delayed spiking (red trace). B, FSI characterized by relatively low input resistance, lack of spontaneous activity, nonlinear spiking in response to depolarizing current pulses, high maximal firing frequency, and large spike afterhyperpolarizations. C, Cholinergic interneurons were characterized by their spontaneous activity (F, top trace and red trace in C) and marked time-dependent sag in response to hyperpolarizing current injections attributable to activation of HCN channels. D, PLTS interneurons exhibited high input resistance and both low-threshold spike and plateau potential (arrow) at the offset of negative current injections. None of these neurons was EGFP+, and all exhibited characteristics identical to those described previously for electrophysiologically and neurochemically identified neurons in striatal slices from rat and mouse. E, Typical responses of a substantia nigra pars compacta (SNc) dopaminergic neuron, fluorescent for EGFP–TH+ (red arrow in inset). EGFP–TH+ nigral dopaminergic neurons exhibit slow regular spontaneous activity (F, bottom trace), long-duration action potentials, and a large time-dependent sag in response to hyperpolarizing current injections attributable to HCN. G, Single action potential from a SPN used to illustrate methods for measurements of action potential threshold (1), action potential duration at half-amplitude (2), afterhyperpolarization (3), and action potential amplitude (4). Scale bar in inset and Figs. 3–6, 20 μm.
Figure 2.
Figure 2.
Four different types of EGFP–TH+ neurons in mouse striatum. A, Selected two-dimensional scatter plots of various electrophysiological parameters reveal the separation of striatal EGFP–TH+ neurons into four distinct groups, termed Types I–IV. AP, Action potential. B, Clustering of four distinct cell types in one representative three-dimensional scatter plot. C, Averaged action potentials from cell Types I–IV clearly show differences in multiple spike waveform parameters. D, Histogram showing the distribution of the four EGFP–TH+ cell types.
Figure 3.
Figure 3.
Electrophysiological properties of Type I EGFP–TH+ neurons. A, Whole-cell current-clamp recordings of responses to negative and positive current pulses in a Type I neuron show high input resistance, strong spike frequency adaptation leading to complete spike failure after small depolarizing pulses (red trace), and the expression of long-lasting plateau potentials (green arrow). Inset, EGFP fluorescent image of recorded neuron and I–V plot showing inward rectification. I–V curves in this and Figures 4–6 are plotted at the end of the current pulse at the point marked by an open circle. B, Some Type I neurons exhibited slow, irregular spontaneous activity characterized by ∼10 mV fluctuations in membrane potential. C, Time-dependent sag in response to hyperpolarizing current injection and rebound slow depolarization after its offset (green arrow) are both blocked by ZD7288 (50 μm), indicating that both are attributable to Ih. D, Rebound bursting after offset of hyperpolarizing current injections. E1, The plateau potential was blocked by 10 μm nimodipine (NIM). E2, Plateau potential in a different Type I neuron is blocked by 100 μm flufenamic acid (FLU), indicating that the plateau potentials are attributable to a Ca2+-activated cation conductance (ICAN). F1, Summary of effects of nimodipine on plateau potential (PP) duration in six Type I neurons. F2, Summary of effects of flufenamic acid on plateau potential (PP) duration in eight Type I neurons. CON, Control. ***p < 0.0001.
Figure 4.
Figure 4.
Electrophysiological properties of Type I and Type II EGFP–TH+ neurons. A, Responses to negative and positive current pulses in a Type II neuron illustrate lower input resistance than Type I neurons, similar time-dependent Ih-like sag in response to hyperpolarizing current injections, and ability to sustain high-frequency firing with little spike frequency adaptation after the first few milliseconds. Inset, EGFP fluorescent image of recorded neuron and I–V plot showing almost linear responses except at most hyperpolarized membrane potentials. B, Example of tonic spontaneous firing. C, D, Electrophysiological properties of Type III EGFP–TH+ neurons. C1, Responses to negative and positive current pulses in a Type III neuron reveal a linear I–V relationship below −90 mV and marked inward rectification at more depolarized potentials. Note the very low input resistance and the very hyperpolarized resting membrane potential compared with Type I and Type II neurons. There is significant spike frequency adaptation, leading to complete spike failure in responses to strongest depolarizing current pulses. C2, When Type III neurons are depolarized by constant current injection, overall excitability is increased, spike frequency adaptation is reduced allowing sustained firing up to 120 Hz, and a prolonged plateau potential appears (green arrow). The plateau potential (D1) is blocked by 10 μm nimodipine (D2), demonstrating the involvement of an L-type Ca2+ channel.
Figure 5.
Figure 5.
Electrophysiological properties of Type IV EGFP–TH+ neurons. A, Responses to negative and positive current pulses in a Type IV neuron show a linear I–V relation over a wide range of membrane potentials. Input resistance is greater than in Type III neurons but less than in Type I and II neurons. B1, Type IV neurons exhibit a short burst of spikes riding on a low-threshold spike at the beginning of the response to depolarizing pulses (asterisk) and rebound burst after the offset of strong hyperpolarizing current pulse as well as a time-dependent sag at hyperpolarized membrane potentials. B2, ZD7288 eliminated both the time-dependent sag in response to hyperpolarizing current injections as well as the burst after relaxation from hyperpolarization, indicating that both are attributable to Ih, the sag to its onset, and the late burst to its offset.
Figure 6.
Figure 6.
Retrograde labeling. A, Medium-magnification fluorescence micrograph of striatum after injections of rhodamine beads into both SN and GP under the EGFP filter. Eleven fluorescent EGFP–TH+ neurons (arrows) are visible. B, Same field as in A but under the rhodamine filter showing numbers fluorescent retrogradely labeled SPNs. C, Merged image of A and B. Note that there is no colocalization of EGFP and rhodamine. D, Rhodamine injection sites in SN and GP for three animals. E, Higher-magnification merged image of the area shown in the white square in C shows that none of the retrogradely labeled neurons express EGFP.
Figure 7.
Figure 7.
Neurochemical identification of striatal EGFP–TH+ interneurons. A–C, Low-magnification double fluorescence micrographs of striatum showing EGFP–TH (green) simultaneously with PV immunofluorescence (A), NOS immunofluorescence (B), and CR immunofluorescence (C). Higher-magnification photomicrographs of the areas outlined in the white boxes are shown in A1–A3 (EGFP–TH, PV, and merged), B1–B3 (EGFP–TH, NOS, and merged), and C1–C3 (EGFP–TH, CR, and merged). Note that none of the EGFP–TH interneurons (white arrows in A1–C1) colocalize PV, NOS, or CR (red arrows). D1–D3, Colocalization of TH immunofluorescence with EGFP–TH 3 d after colchicine injection. D1, EGFP–TH+ neurons (white arrows). D2, Immunoreactive TH+ neurons (red arrows). D3, Merge of D1 and D2 showing neurons expressing both EGFP and TH (yellow arrows), EGFP but not TH (green arrow), and TH but not EGFP (red arrow). Str, Striatum; Ctx, cortex.
Figure 8.
Figure 8.
Results of single-cell RT-PCR performed on four EGFP–TH+ neurons. A, Typical responses of a Type I neuron to depolarizing current injection. B, Agarose gel from the cell shown in A plus another Type I neuron showing that both express the amplicon for VMAT1 but not VMAT2. C, Typical responses of a Type IV neuron to depolarizing current injection. D, Gels from the neuron shown in C plus a second Type IV neuron show that Type IV neurons also express VMAT1. MW, Molecular weight.
Figure 9.
Figure 9.
Synaptic input to EGFP–TH+ neurons. A, Experimental design. Bipolar stimulating electrodes were placed onto the surface of the deep layers of the cortex (1) and in striatum to evoke responses in striatal EGFP–TH+ neurons. CPu, Caudate putamen. B1, In a representative Type I cell, local stimulation evoked a biphasic response consisting of an overlapping EPSP and IPSP (control). B2, DNQX (10 μm) blocked the EPSP component, leaving a pure IPSP with an apparent reversal potential of −65 mV (B4). B3, The IPSP was blocked by bicuculline (10 μm), indicating mediation by GABAA receptors. C, Same experiment performed with a Type II neuron but reversing the order of the antagonist application. C1, Local stimulation evokes biphasic response. C2, The inhibitory component was blocked by 10 μm bicuculline, showing that it was mediated by a GABAA receptor. C3, The remaining EPSP is completely eliminated by addition of DNQX (10 μm). D1, Cortical stimulation evokes short-latency monosynaptic DPSP in a Type I neuron. D2, The DPSP is not affected by 10 μm bicuculline. D3, The AMPA/kainate channel blocker DNQX (10 μm) completely eliminates the DPSP, showing that it is a glutamatergic EPSP. D4, Time course of drug effects on the EPSP.
Figure 10.
Figure 10.
Paired whole-cell current-clamp recording between a presynaptic Type I neuron and a postsynaptic SPN. Both neurons were recorded with a CsMeSO4 internal solution. A, The Type I neuron (red arrow, pre) is fluorescent and shows a typical response to depolarizing current (inset). The postsynaptic SPN (post) is not fluorescent and cannot be seen under epifluorescence illumination. Scale bar, 20 μm. B, Depolarizing the SPN to −40 mV reveals both spontaneous EPSPs (green arrowheads) and IPSPs (red arrows). C1, Current pulses in the Type I neuron elicit three spikes that evoke three hyperpolarizing IPSPs in the SPN at −40 mV that summate to >1 mV. C2, The IPSP was reversibly blocked by bicuculline (10 μm) and recovered after wash (C3), showing that the synaptic response was mediated by a GABAA receptor.
Figure 11.
Figure 11.
Spiking in Type II and Type III neurons elicits IPSCs in SPNs and delays firing in response to current injection. A, Connected pair consisting of a presynaptic EFGP+ Type II neuron (top left) and a postsynaptic SPN (bottom left). The insets are high-magnification micrographs showing an aspiny Type II (pre) dendrite and a spiny SPN dendrite (post) after both were patched in whole-cell mode and stained with Alexa 594. B, Top, Current pulses injected into the Type II neuron elicited a train of presynaptic spikes that evoked IPSCs in the postsynaptic SPN. Both short-term depression and early facilitation were evident. C, Two spikes evoked by two 5 ms pulses 50 ms apart in the Type II neuron result in modest paired-pulse facilitation in the same SPN. Black traces are averages, and gray traces are individual trials. D, The IPSCs were reversibly blocked by bicuculline (10 μm) showing that they were mediated by a GABAA receptor. E, Connected pair consisting of a presynaptic Type III neuron and a postsynaptic SPN (inset). Two presynaptic spikes separated by 50 ms (bottom trace) delay the occurrence of depolarization induced spiking in the postsynaptic SPN (top black trace) by almost 100 ms (top red trace). F, Entire dataset from which the traces in E were selected. Depolarization evoked spiking in SPN under control conditions (black traces) and in the presence of two spikes in the presynaptic neuron (red traces). Note the reliability of this connection. G, I–V plot for this synaptic response showing a reversal potential consistent with mediation by Cl ions and a synaptic conductance near 1 nS.
Figure 12.
Figure 12.
Whole-cell recordings of two connected pairs in which the presynaptic neuron was an SPN and the postsynaptic neuron was a Type I neuron. A–D, Presynaptic and postsynaptic neurons recorded with 140 mm CsMeSO4 internal solution. A, Postsynaptic EGFP–TH+ Type I neuron (red arrow, post) is fluorescent and shows typical response to depolarizing current pulses. Presynaptic SPN are not fluorescent and cannot be seen (pre) and were patched under DIC visualization. B1, Depolarizing current pulse evokes three spikes in a Type I neuron (top black trace). B2, Single action potential elicited in the SPN (red trace) causes an IPSP (−45 mV) in the Type I neuron held at −48 mV (black trace) that was sufficient to completely block the second of three spikes evoked in the Type I neuron. Mean IPSP amplitude, 0.58 ± 0.14 mV; latency, 2.6 ± 0.13 ms; p = 100%. C, Single spike (top red trace) in the SPN evokes a hyperpolarizing IPSP in a Type I neuron held at −46 mV. Black trace is average, and gray traces are individual trials. Note that the IPSP failure rate is zero. The IPSP is reversed at −80 mV (middle) and is completely blocked by 10 μm bicuculline (bottom), indicating a GABAA IPSP. D, Comparison of a spontaneous IPSP and one evoked from the SPN shows them to have identical amplitudes and time courses, suggesting that some of the spontaneous IPSPs in Type I neurons arise from surrounding SPNs. E–G, Another connected pair in which the presynaptic neuron was an SPN and the postsynaptic neuron was a Type I. Both presynaptic and postsynaptic neurons recorded with normal internal solution. E, Postsynaptic Type I neuron (red arrow, post) is fluorescent. Presynaptic SPN is not fluorescent and cannot be seen (pre) and was patched under DIC visualization. F, Train of spikes in the presynaptic SPN elicited by a current pulse (red traces) elicit IPSPs (green arrows) in the postsynaptic Type I neuron (black traces). The IPSPs delay the onset of the subsequent spike. G, Single depolarization-evoked spikes in the SPN elicit IPSPs that delay the occurrence of the subsequent spike in the postsynaptic Type I neuron depolarized to threshold by current injection. H2, Bicuculline at 10 μm blocks the IPSPs and the subsequent postsynaptic spikes occur with a decreased latency. H3, The IPSP returns after washout, and the latency to the subsequent postsynaptic spike increases. Scale bars: A, E, 20 μm.
Figure 13.
Figure 13.
Drawing tube reconstructions of EGFP–TH+ neurons filled with biocytin after recording, superimposed on concentric circles for Sholl analyses. Striatal EGFP–TH+ neurons had three to five primary dendrites that branched between 15 and 50 μm from the soma and gave rise to a modest arborization of secondary and tertiary varicose dendrites. Dendrites are plotted in black and axons in red in the Sholl plots. The quantitative morphometric results are given in Table 3. All Type II–IV EGFP–TH+ neurons were aspiny, but 6 of 26 Type I neurons expressed sparse spine-like processes on their higher-order dendrites (e.g., A and C; see Fig. 14). The axonal arborizations were sometimes quite dense (A–C), reaching a maximum density between 50 and 120 μm from the soma (red circles in Sholl plots) and were studded throughout with small varicosities (A–D; deep red dots on the axons), presumably synaptic boutons. In these reconstructions, the soma and dendrites are shown in black, and the axon are shown in red and varicosities in dark red.
Figure 14.
Figure 14.
Characteristic anatomy of a typical SPN and a spiny EGFP–TH+ Type I neuron filled with biocytin after whole-cell recording from adult mouse slices. A, Low-magnification photomicrograph of an SPN shows a soma ∼12 μm in diameter that gives rise to six primary dendrites that are aspiny for the initial 20 μm that then become densely invested with spines and give rise to secondary and higher-order spiny dendrites extending for a radius of ∼300 μm. 1–5, Higher-magnification images of spiny dendrites (A2–A5) and axons (A1, A2) from regions marked with the same numbers in A above. Note the high density of dendritic spines. The mushroom-like appearance of many of the spines is particularly apparent in A4 and A5. The local axon collaterals are varicose and exhibit intermittent varicosities. A6, Whole-cell current-clamp recording of responses to intracellularly injected current pulses from this neuron is shown in 6 and is very characteristic of SPNs. B, A spiny Type I neuron shows a slightly larger soma and four thick primary dendrites that branch less frequently and follow a straighter course than the dendrites of an SPN. The dendrites are invested with spine-like appendages. These spine-like processes are of much lower density than the dendritic spines on the SPN, many of them are quite long (arrows in B), and most appear to lack spine heads (B2). Like the SPN, the axonal arborization of the Type I neuron is quite dense. The intervaricose segments appear thinner than those of the SPN, and the entire axonal arborization is studded with larger and more prominent varicosities than that of the SPN (B1). B3, The whole-cell current-clamp recording from this neuron is typical for Type I neurons and completely distinct from that of a typical SPN.

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