Heterogeneous properties of central lateral and parafascicular thalamic synapses in the mouse striatum

T. Ellender, J. Harwood*, P. Kossilo*, P. Bolam

* contributed equally.

Anatomical Neuropharmacology Unit

Keywords: thalamus, striatum, intralaminar, parafascicular, central lateral

Abbreviated title: Properties of thalamic afferents of MSNs

Corresponding author:

T. Allender

Anatomical Neuropharmacology Unit, University of Oxford

t.allender@dpag.ox.ac.uk

Abstract

To understand the principles of operation of the striatum it is critical to elucidate the properties of the main excitatory inputs from cortex and thalamus, and their ability to activate the principal neurons of the striatum, the medium spiny neurons (MSNs). The thalamostriatal projection is heterogeneous and we aimed to study these afferent inputs to MSNs using small localized injections of adeno associated virus carrying fusion genes for channelrhodopsin-2 and YFP, in either the rostral or caudal portions of the intralaminar thalamic nuclei (i.e. the central lateral or parafascicular nucleus) in mice. This enabled optical activation of specific thalamic afferents combined with whole-cell, patch-clamp recordings of MSNs and simultaneous electrical stimulation of cortical afferents, in adult mice.

We found that the subtypes of thalamostriatal synapses differ in their basic properties, short-term dynamics and expression of ionotropic glutamate receptor subtypes. Our results suggest that central lateral synapses are most efficient in driving MSNs, particularly those of the direct pathway, to depolarization as they exhibit large amplitude responses, short-term facilitation and predominantly express postsynaptic AMPA receptors. In contrast, parafascicular synapses exhibit small amplitude responses, short-term depression and predominantly express postsynaptic NMDA receptors suggesting a modulatory role, e.g. facilitating Ca ²⁺-dependent processes. Indeed, pairing parafascicular, but not central lateral, presynaptic stimulation with action potentials in MSNs, leads to NMDA receptor- and Ca ²⁺-dependent long term depression at these synapses. We conclude that the main excitatory thalamostriatal afferents differ in many of their characteristics and suggest that they each contribute differentially to striatal information processing.

Introduction

The basal ganglia are a group of subcortical brain nuclei essential for the control of movement and a variety of other functions (Graybiel et al., 1994; Grillner et al., 2005; Yin and Knowlton, 2006). The striatum is the main input nucleus of the basal ganglia receiving excitatory inputs from the cortex and the thalamus (Buchwald et al., 1973; Smith et al., 2004; Doig et al., 2010). The main thalamic afferents to the striatum originate in the intralaminar nuclei (Macchi et al., 1984), which in rodents, can be divided into the rostral central lateral (CL) and the caudal parafascicular (Pf) nucleus (Berendse and Groenewegen, 1990; Castle et al., 2005; Smith et al., 2009). Both thalamic projections make direct synaptic contacts with the main classes of medium-sized spiny neuron (Xu et al., 1991; Sadikot et al., 1992; Lacey et al., 2007). The Pf afferents furthermore, make contact with several types of striatal interneuron (Lapper and Bolam, 1992; Rudkin and Sadikot, 1999; Sidibe and Smith, 1999).

Although the thalamostriatal pathway gives rise to similar numbers of synapses on MSNs as does the corticostriatal pathway (Lacey et al., 2005; Fujiyama et al., 2006; Raju et al., 2006; Moss and Bolam, 2008; Doig et al., 2010), the properties of thalamostriatal synapses have proven difficult to study because of the heterogeneity of the projection and the trajectory of the axons connecting the thalamus and striatum. The latter difficulty can be overcome, to some extent, when studying the projection as a single entity, by careful placement of stimulating electrodes and careful selection of the plane of slicing of the brain (Ding et al., 2008; Smeal et al., 2008). However, electrical stimulation cannot isolate different sub-nuclei of the thalamostriatal system. It is clear that different sub-nuclei have different properties; for instance, it has been shown that thalamostriatal neurons in the CL and Pf nuclei differ in their morphology, firing properties, as well as their striatal targets (Lacey et al., 2007), presumably underlying different roles in striatal function. Furthermore, behavioral studies, mainly focused on Pf, have suggested thalamic involvement in a variety of processes (Kendall et al., 2005).

The aim of the work described in this paper was to test the hypothesis that synapses formed in the striatum by neurons originating in different sub-nuclei of the intralaminar thalamus have different functional properties. To address this we set out to isolate and differentially activate the thalamostriatal projection originating in either the rostral or caudal portion of the intralaminar nuclei using precise viral delivery of channelrhodopsin-2 (ChR2) (Boyden et al., 2005). Whole-cell patch-clamp recordings of neurochemically identified MSNs with optical activation of these specific thalamic inputs enabled us to identify the properties of the two types of thalamic synapse, as well as electrically activated cortical synapse in the adult mouse striatum. We conclude that thalamostriatal synapses with striatal principal neurons show a hitherto unknown heterogeneity which suggests that they subserve different roles in striatal computation.

Methods and Materials

Animals. All experiments were carried out on transgenic mouse lines which were bred and housed in accordance with the Animals (Scientific Procedures Act) and the Society of Neuroscience policy on the use of animals in neuroscience research. Recordings were carried out on CAMKII-cre mice which express cre recombinase in all CAMKII expressing neurons and were obtained from Jackson laboratory and kept as a homozygous breeding line.

Viral transfection. Adeno associated virus serotype 2 (AAV2) carrying fusion genes for ChR2 / ChETA and YFP were injected into thalamus of CAMKII-cre mice between postnatal day 14 and 21. Either the rostral intralaminar nuclei (mostly CL) or caudal intralaminar nuclei (mostly Pf) were targeted. Typical coordinates from Bregma for rostral intralaminar nuclei injections were lateral, 0.6 mm; posterior, 1.4 mm; and 3.1 mm depth from surface of brain. Typical coordinates from Bregma for caudal intralaminar nuclei injections were lateral, 0.7 mm; posterior, 2.4 mm; and 3.5 mm depth from surface of brain. Viral DNA included the double-floxed sequence for ChR2 (H134R)-EYFP driven by the elongation factor 1 promotor or the double floxed sequence for ChR2 (E123T-H134R)-EYFP driven by the elongation factor 1 promotor. AAV2 particles were produced at the University of North Carolina Gene Therepy Center Virus Vector Core. Typical titers were ~10 ¹² IU/ml. Injection volumes were 300 nl. After allowing 6 - 12 weeks for ChR2 or ChETA expression, acute striatal slices were prepared.

Slice preparation. Oblique coronal striatal slices (300 - 400 um) were prepared from 2 to 4-month old injected CAMKII-cre mice. Mice were anesthetized with isoflurane and decapitated. Slices were prepared in artificial cerebrospinal fluid (ACSF) containing (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH ₂ PO ₄ , 5 MgCl ₂ , 2.5 CaCl ₂ , 24 NaHCO ₃ , and 10 glucose, pH 7.2-7.4, bubbled with carbogen gas (95% O ₂ / 5% CO ₂ ). Slices were immediately transferred to a storage chamber containing ACSF (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH ₂ PO ₄ , 1.5 MgCl ₂ , 2.5 CaCl ₂ , 24 NaHCO ₃ , and 10 glucose, pH 7.2-7.4, bubbled with carbogen gas, at 37deg C for 30 min and subsequently maintained at room temperature until used for recording.

Recording. Whole-cell current-clamp and voltage-clamp recordings from dorsal striatal MSNs were performed using glass pipettes, pulled from standard wall borosilicate glass capillaries containing, for whole-cell current-clamp (in mM): 110 potassium gluconate, 40 HEPES, 2 ATP-Mg, 0.3 Na-GTP, 4 NaCl and 4 mg/ml biocytin (pH 7.2-7.3; osmolarity, 290-300 mosmol/l); and for whole-cell voltage-clamp (in mM): 120 cesium gluconate, 40 HEPES, 4 NaCl, 2 ATP-Mg, 0.3 Na-GTP, 0.2 QX-314 and 4 mg/ml biocytin (pH 7.2-7.3; osmolarity, 290-300 mosmol/l). In the plasticity experiments either EGTA (10 mM) or MK-801 (1 mM) was included in the current-clamp internal solution. All recordings were made using an EPC9/2 HEKA amplifier with integrated A/D converter and acquired using Pulse software (HEKA Electronik).

Stimulation and recording protocols. MSN afferents were stimulated electrically and optically. Electrical stimulation was performed by placing an ACSF filled glass electrode in the external capsule for activation of corticostriatal afferents. Stimulation strength was set to evoke approximately 1/3 of maximum response corresponding to a stimulation strength of 100-300 uA. Optical stimulation of thalamostriatal afferents was performed using the optoLED system (Cairn Research), consisting of a 470 nm, 3.5 W LED mounted on a Zeiss Axioskop 2 FS microscope, to give 3 ms duration light pulses of ~5% of maximum output power. The spot size corresponded to the area of the slice visualized using a 40x/0.8NA water immersion objective. Activation of excitatory afferents was performed in the presence of blockers of inhibitory GABAergic transmission including the GABA _A -receptor antagonist SR95331 (10 uM) and GABAB-receptor antagonist CGP52432 (2 uM). Fibers were activated every 10 sec and excitatory postsynaptic currents (EPSCs) or excitatory postsynaptic potentials (EPSPs) were recorded in the patched MSN. Evoked EPSCs were recorded in whole-cell voltage-clamp mode at a holding potential near -80 mV and evoked EPSPs in whole-cell current-clamp mode at resting membrane potential. For paired-pulse stimulation, two stimulating pulses were consecutively given at 50 ms interval and repeated every 10 sec for up to 20 times. Trains of pulses consisted of 9 pulses at 5, 10 and 20 Hz, with the latter two followed by a recovery pulse 500 ms later, and was repeated every 30 sec for up to 5 times. Combined AMPA and NMDA receptor-mediated currents were recorded from MSNs held at +45 mV. AMPA receptor-mediated-currents were recorded after 5 min wash-in of d-AP5 (50 uM). Both consisted of at least 20 evoked responses at 0.1 Hz. Pairing induced plasticity protocol consisted of pairing presynaptic activation of thalamic afferents with a single postsynaptic action potential in MSNs, induced by a suprathreshold current step (1 nA, 10 ms), with approximately 8 ms delay. After a 5 min baseline recording of EPSPs at 0.1 Hz the pairing protocol was repeated 100 times at 1 Hz. Subsequently EPSPs were recorded for 25 min at 0.1 Hz.

Analysis of intracellular recordings. Data was analyzed offline using custom written procedures in Igor Pro (Wavemetrics). Postsynaptic currents (PSCs) were detected as upward or downward deflections of more than 2 standard deviations (SD) above baseline. Paired-pulse ratios were calculated by dividing the average slope of the second PSC (S2) with the average slope of the first PSC (S1). Slopes of individual PSCs were determined between 20% - 80% of maximum PSC amplitude. Trains were analyzed by taking the amplitude of each PSC and dividing this by the amplitude of the first PSC. The analysis of EPSC kinetics (peak amplitude, duration, rise time (between 20-80% from peak) and decay time) was performed on individual synaptic responses. NMDA / AMPA ratios were calculated from an average trace of combined AMPA and NMDA receptor-mediated currents and AMPA receptor-mediated currents. The average AMPA receptor-mediated current trace was subtracted from the combined AMPA and NMDA receptor-mediated current trace to obtain the NMDA receptor-mediated current. The maximum amplitude of the NMDA receptor-mediated current was divided by the maximum amplitude of the AMPA receptor-mediated current to obtain the NMDA / AMPA ratio. In plasticity experiments the average amplitude of every two EPSPs are plotted. Plastic changes at synapses were assessed by comparing the average amplitude of recorded EPSPs during the last minute of baseline and the last minute of post induction recording.

Histological analyses. Following intracellular recording, the slices were fixed overnight in 4% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (PB; pH 7.4) at 4degC. After washes the slices were embedded in 5% agar and resectioned at 50 um on a vibrating microtome (VT1000S; Leica Microsystems). All sections were preincubated in 10%-20% normal donkey serum (NDS; Vector Laboratories) in PBS for more than 1 h at room temperature. Biocytin-filled cells were visualized by incubating sections in 1:10,000 streptavidin-405 conjugate (Invitrogen) in PBS containing 0.3% Triton-X (PBS-Tx) overnight at 4degC. YFP expression of thalamic fibres was visualized by incubating sections in 1:1000 chicken anti-GFP (Aves Labs) in PBS-Tx and 1% NDS overnight at 4degC followed by 1:500 donkey-anti-chicken-488 fluophore (Jackson Immunoresearch Laboratories) in PBS-Tx for 2 h at room temperature. To define the subtype of MSN in recordings performed, the sections were heated at 80degC in 10 mM sodium citrate (pH 6.0) for approximately 30 min prior to incubation with 1:1000 rabbit anti-preproenkephalin (LifeSpan biotechnology) in PBS-Tx and 1% NDS overnight at 4degC after which the reaction was revealed by incubating with 1:500 donkey-anti-rabbit-Cy3 fluophore (Jackson Immunoresearch Laboratories) in PBS-Tx for 2 h at room temperature. The neurons that were immunopositive were classified as indirect pathway MSNs.

To confirm the AAV injection was correctly localized in the thalamic intralaminar nuclei, following preparation of the slices for recording, the remainder of the brain, containing thalamus, was immersion fixed overninght in 4% paraformaldehyde and 15% saturated picric acid in 0.1 M phosphate buffer (PB; pH 7.4) at 4degC. After washes the brain blocks were resectioned at 50 um on a vibrating microtome (VT1000S; Leica Microsystems). The sections were incubated in 20% NDS (Vector Laboratories) for 45 min at room temperature. YFP was visualised by immunolabeling using 1:1000 chicken-anti-GFP (Aves Labs) in PBS-Tx and 1% NDS overnight at 4degC followed by 1:500 donkey-anti-chicken-488 fluophore (Jackson Immunoresearch Laboratories https://www.jacksonimmuno.com) in PBS-Tx for 2 h at room temperature. To facilitate anatomical characterization, the sections were incubated for 30 min in a 1:200 Nissl-Cy5 stain (Neurotrace, Invitrogen). Finally, all sections were mounted in Vectashield (Vector Laboratories) and images were captured with a LSM 710 (Zeiss, Gottingen, Germany) confocal microscope using ZEN and Axiovision software (Zeiss, Gottingen, Germany). The software's default settings for fluorophores were used for beamsplitters and ranges of emissions sampled.

Statistics. All data are presented as means +- SEM, except where stated. Student's t tests and repeated measures ANOVA were performed using SPSS 17.0 (* p<0.05, ** p<0.01). P values are given with 95% confidence intervals.

Drugs and Chemicals. All drugs were obtained from Tocris Biosciences (Bristol, UK) and Sigma-Aldrich (Poole, Dorset, UK).

Results

Localized transfection of CL or Pf thalamic neurons

The main thalamic input to the striatum comes from the rostral (central lateral) and caudal (parafascicular) portion of the intralaminar nuclei (Macchi et al., 1984), which have distinct morphological and electrophysiological properties (Lacey et al., 2007). We set out to characterize these different inputs to MSNs by using small volume (300 nl) injections of AAV2 containing the double-floxed sequence for the light activatable channel ChR2 or ChETA in either the rostral or caudal portions of the intralaminar nuclei of CAMKII-cre mice (Figure 1A, B). We were able to inject either nucleus with minimal overlap, as assessed by the localization of YFP-expressing neurons (Figure 1C, D), indicating that transfection was localized to either the CL or Pf subnuclei of the intralaminar thalamus. All other photographed transfections can be found in supplementary materials 1.

MSNs receive both thalamic input and cortical input

Following a survival time of 6 - 12 weeks, brain slices of the forebrain were prepared from injected CAMKII-cre mice. The relatively long survival time enables sufficient expression of ChR2 or ChETA throughout the axonal arbor of the thalamic neurons including their projections to the striatum. Fluorescence illumination of the striatum in these brain slices reveals a dense network of YFP-positive fibers (Figure 2B). We also observed YFP-positive fibers in the cortex asa consequence of the expression of ChR2-YFP in their axonal projections to the cortex (Krettek and Price, 1977; Herkenham, 1979; Groenewegen, 1988). Whole-cell, voltage-clamp and current-clamp recordings of striatal MSNs were performed while activating thalamic afferents by illuminating the striatum with brief light pulses (470 nm; 3 ms duration), as well as cortical afferents by electrical stimulation in the external capsule (Figure 2A). All the experiments were performed in the presence of the GABA _A -receptor antagonist SR95331 (10 uM) and the GABA _B -receptor antagonist CGP52432 (2 uM). The subtype of MSN was confirmed post hoc by immunolabeling for preproenkephalin (PPE) which is selectively expressed by indirect pathway MSNs (Figure 2C, D). If a recorded neuron was negative for PPE and was defined as an MSN on the basis of the somatodendritic morphology, it was considered a direct pathway MSN. A total of 117 MSNs were recorded, of which 35 (30%) were unequivocally identified as PPE-immunonegative (PPE-) and 37 (32%) as PPE-immunopositive (PPE+). Cortical and thalamic responses were recorded in both PPE- and PPE+ MSNs (Figure 2E, F).

Basic synaptic properties of thalamic synapses on MSNs

We first set out to study the minimal optical stimulation strength necessary to elicit a thalamic response in approximately half of the stimulations. We found that with LED stimulation strength as low as ~35 mW we could elicit responses in striatal MSNs from both CL and Pf afferents when recording in whole-cell, voltage-clamp mode. We found that the EPSC properties for CL and Pf synapses using this stimulation strength are remarkably similar, with some small differences in that CL EPSCs were larger in amplitude (t = 2.31, p<0.05) and Pf EPSCs slightly longer in duration (t= 1.01, p=0.02). In all subsequent experiments we used higher stimulation strength to elicit approximately half maximum amplitude responses. The average amplitude of response following CL axon stimulation was 66.3 +- 13.5 pA (stim strength; 450 mW; n = 16), whereas those following Pf axon stimulation were significantly smaller at 30.7 +- 4.1 pA (stim strength: 980 mW; t = 0.81, p<0.05). The remaining EPSC characteristics (duration, rise time and decay time) were not significantly different between CL and Pf inputs (Table 1).

We further characterized the properties of EPSCs elicited by photoactivation of CL or Pf afferents by defining the postsynaptic MSN as a direct or indirect pathway neuron by the expression or not of PPE. This analysis revealed that the response to CL axon stimulation was consistently larger in amplitude than the response to Pf for both direct and indirect pathway MSNs (direct pathway: t(27) = 1.98, p<0.05). Furthermore, the analysis revealed that the response to CL axon stimulation was larger in amplitude on direct pathway neurons (direct pathway: t(27) = 1.99, p<0.05; Table 1).

These results suggest, first, that thalamic synapses from CL produce a larger depolarization of both direct and indirect pathway MSNs than Pf synapses. Secondly, CL synapses with direct pathway MSNs produce a stronger response those with indirect pathway MSNs.

Short-term plastic properties of thalamic and cortical synapses on MSNs

We next set out to investigate the dynamic properties of the excitatory synapses from CL, Pf and cortex. MSNs were recorded in whole-cell, voltage-clamp and current-clamp mode while thalamic and cortical afferents were activated using a paired-pulse stimulation protocol consisting of two pulses at 50 ms interval. We found that the paired-pulse ratio (PPR) for afferents from CL are facilitating (t(55) = 2.02, p<0.05), whereas those from Pf are depressing (t(27) = 3.12, p<0.05). The variation in paired-pulse ratio between individual MSNs leads to an average 'neutral' PPR in the case of cortical afferents (mean ratio of S2/S1: 1.00 +- 0.04; n = 54; Figure 3A). We did not observe any differences in the PPR of CL, Pf and cortical synapses between direct pathway and indirect pathway MSNs (Table 1).

Next, we investigated the dynamics of these inputs during longer trains of stimulation at 5 Hz, 10 Hz and 20 Hz. These analyses revealed a similar picture to that seen during paired-pulse stimulation. Whereas all responses depress eventually, only the responses of CL inputs are facilitating for the first few spikes during both 10 and 20 Hz stimulation CL (t(72) = 1.73, p<0.05; Figure 3B). Secondly, the cortical response recovers more quickly than the thalamic response after both 10 Hz and 20 Hz stimulation (ratio of response to first pulse: 10 Hz; cortical 0.87 +- 0.05, vs. thalamic 0.57 +- 0.05, 20 Hz; cortical, 0.90 +- 0.12 vs. t(72) = 3.07, p<0.05). The Pf response is significantly depressing following both the 10 and 20 Hz trains and the rate of recovery was the least of all three responses (t(44) = 3.10, p<0.05; Figure 3B). Thus the dynamic properties of CL, Pf and cortical synapses differ in that CL synapses are facilitating whereas Pf and cortical synapses are largely depressing, suggesting that CL inputs are well-suited to drive MSN depolarization during trains or bursts of action potentials.

Dominant ionotropic glutamate receptors at thalamic and cortical synapses

We found differences between the presynaptic short-term dynamics of CL and Pf synapses but it is equally possible that there are postsynaptic differences. We therefore, investigated the ionotropic glutamate receptor composition postsynaptic to CL, Pf and cortical synapses.

Stimulated excitatory events were recorded from MSNs held at +40 mV which were the result of both NMDA and AMPA receptor-mediated currents. After baseline recording, d-AP5 (50 uM) was bath applied to block the NMDA receptor-mediated currents, thereby isolating the AMPA receptor-mediated component. We found that CL synapses are dominated by AMPA receptor-mediated currents, whereas Pf synapses are dominated by NMDA receptor-mediated currents (t(27) = 5.31, p<0.01). Cortical synapses exhibit an NMDA/AMPA ratio in between those of CL and Pf synapses and dominated by NMDA receptor-mediated currents (mean NMDA/AMPA ratio for cortical: t(27) = 2.66, p<0.05). These results suggest that input from CL is very efficient at depolarizing MSNs whereas inputs from Pf would have little effect on MSNs at resting membrane potential.

Long-term plasticity at Pf, but not CL, synapses

These results suggest that NMDA receptors gate the dominant excitatory postsynaptic current at Pf synapses. This would enable this synapse to gate substantial amounts of Ca ²⁺ if appropriately activated, and this could facilitate Ca ²⁺-dependent processes including some forms of synaptic plasticity. We therefore investigated the ability of CL and Pf synapses to undergo pairing induced plastic changes by performing whole-cell, patch-clamp recordings from MSNs in current clamp mode with optical stimulation of thalamic afferents. We recorded baseline EPSPs for 5 min, after which we used a pairing protocol consisting of an optically evoked thalamic EPSP followed by an action potential in the MSN evoked by suprathreshold current injection (delay ~8 ms). We investigated the effect of this pairing protocol on the efficacy of the thalamic input. We found that this protocol leads to long-term depression of evoked EPSPs at Pf. However, the same protocol applied to CL synapses did not lead to any change in the amplitude of evoked EPSPs (t(27) = 0.41, p<0.05; Figure 5A).

To investigate the mechanisms underlying this plasticity we repeated the pairing protocol for Pf synapses, but this time included either the calcium chelator, EGTA (10 mM), or the NMDA receptor antagonist, MK-801 (1 mM), in the intracellular solution. We waited an additional 5 min before starting the experiment to ensure sufficient diffusion of these compounds throughout the recorded MSN. The presence of MK-801 (Figure 5C) or EGTA (Figure 5D) blocked the induction of long-term depression at Pf synapses. These results thus suggest that Pf synapses exhibit long term plasticity that is postsynaptically mediated by calcium flowing through NMDA receptors.

Discussion

The main findings of the present study are that thalamostriatal synapses with MSNs originating in different parts of the intralaminar thalamus have markedly different properties. Using an optogenetic approach in mice we were able to dissociate between the rostral (CL) and caudal (Pf) intralaminar inputs to MSNs. First, we find that CL synapses give rise to larger amplitude responses than Pf synapses. Secondly, CL synapses are facilitating, whereas Pf synapses are depressing. Thirdly, CL synapses predominantly express postsynaptic AMPA receptors, whereas Pf synapses are predominantly associated with postsynaptic NMDA receptors. Finally, the relatively high expression of NMDA receptors at Pf synapses facilitates pairing-induced plasticity at these, but not CL, synapses. Corticostriatal synapses exhibited properties in between those of CL and Pf synapses. These findings suggest that CL and Pf inputs subserve different roles in striatal computation.

Properties of thalamostriatal synapses on MSNs

We used optogenetic tools to isolate specific thalamic nuclei and their projections to MSNs by injections of AAV particles containing the double floxed sequence for ChR2 in either the rostral or caudal portion of the intralaminar nuclei of CAMKII cre mice. This approach leads to the expression of ChR2 in excitatory thalamic neurons around the site of injection including their axonal projections to the striatum. We were thus able to selectively stimulate and dissect out the properties of CL and Pf afferents in slices of striatum, tasks that are not possible using conventional electrical stimulation in slices or indeed, in vivo. Furthermore, we used mice at 2 to 3 months of age that enabled us to investigate the properties of the thalamic synapses in the adult thalamostriatal circuits. We also examined cortical synapses with MSNs by electrical stimulation of the external capsule to define their properties in adult animals and to compare them to thalamostriatal synapses. We did not find any significant changes in thalamostriatal axons in either their morphology or ability to be activated after this long period of expression of ChR2-YFP (Ellender et al., 2011).

Using this approach we demonstrated that MSNs giving rise to the direct pathway and those giving rise to the indirect pathway (identified by the failure to express or the expression of PPE immunoreactivity, respectively) both receive input from Pf or CL. Furthermore, concurrent electrical stimulation of cortical afferents revealed that MSNs that responded to stimulation of thalamic fibers also receive input from the cortex. Previous electrophysiological (Kitai et al., 1976; Ding et al., 2010) and anatomical studies (Doig et al., 2010) have shown convergence of cortical and thalamic input at the level of individual MSNs, the present results now demonstrate that individual direct and indirect pathway MSNs receiving cortical input may also receive input from CL or Pf.

Analysis of the basic synaptic properties of CL and Pf synapses using minimal and high stimulation strengths revealed similar characteristics between the two types of synapse in terms of rise time, duration and decay time. However, the analysis revealed that stimulation of CL afferents led to larger amplitude postsynaptic events than stimulation of Pf afferents in both voltage-clamp and current-clamp recordings. Although differences in density of afferents, release probability and density of postsynaptic receptors will affect this, our analyses of receptor expression (see below) suggest that differences in the dominant postsynaptic glutamatergic receptors at these synapses most likely also play a role. The short-term dynamic responses of the synapses revealed by paired-pulse and train stimulation also revealed very different properties of CL and Pf synapses. Although stimulation at 5 Hz did not lead to changed dynamics, stimulation at 10 Hz led all three excitatory inputs (CL, Pf and cortical) to eventually depress. However, CL synapses exhibited a prominent short-term facilitation prior to depression. This was even more prominent during 20 Hz stimulation; while Pf and cortical synapses depress with every subsequent spike, CL synapses are facilitated for the first few spikes. Previous studies showed short-term depression (Ding et al., 2008) or facilitation (Smeal et al., 2007) of thalamostriatal synapses, when the thalamostriatal pathways are examined as a single entity using electrical stimulation, which suggests a preferential activation of certain afferents dependent on electrode placement, although an age-dependent difference is also possible. Since CL neurons give rise to LTS bursts that can reach a firing frequency of up to 250 Hz (Lacey et al., 2007), it is critical to know whether similar dynamics hold true at this spike frequency. We attempted to investigate this using ChETA, a light-activated channel that can respond to much higher frequencies of stimulation than ChR2 (Gunaydin et al., 2010). However, we did not get reliable responses during stimulation suggesting the single channel conductance of the channel or expression, although sufficient for somatic activation, might be too low to elicit reliable transmitter release from axons (Berndt et al., 2011). Nevertheless, our data, together with the observation that CL synapses predominantly express AMPA receptors, suggest that small bursts of action potentials arriving from CL would be faithfully transmitted and are likely to be very efficient at depolarizing MSNs. This is consistent with the finding that CM stimulation in monkeys consistently leads to an increase in firing of MSNs (Nanda et al., 2009). In contrast to CL synapses, Pf synapses exhibit short-term depression and mainly express postsynaptic NMDA receptors suggesting that these synapses do not lead to strong depolarization of MSNs at resting membrane potential when the NMDA receptor is blocked by Mg ²⁺. However, the expression of postsynaptic NMDA receptors may lead to sufficient Ca ²⁺ influx, under the right conditions, facilitate Ca ²⁺-dependent processes, including state transitions (Plotkin et al., 2011), local dendritic spike modulation (Losonczy et al., 2008) or synaptic plasticity (Kreitzer and Malenka, 2008).

We have previously shown that the properties of corticostriatal synapses in striatal slices of young mice are essentially indistinguishable whether examined by electrical stimulation or optogenetically (Ellender et al., 2011), we were thus confident to compare the properties of corticostriatal synapses with those of thalamostriatal synapses in the present study. We found that the properties of cortical synapses in the adult striatum lie in between those of CL and Pf synapses. Electrical stimulation of the external capsule leads to large amplitude responses in both direct and indirect pathway MSNs. The paired-pulse and train stimulation experiments showed that cortical synapses are mostly depressing (Smeal et al., 2007). We did not find the short-term facilitation seen in slices from younger animals (Ding et al., 2008; Ellender et al., 2011), suggesting this might be an age-dependent phenomenon. Lastly, we found that cortical synapses with MSNs express a high proportion of NMDA receptors (NMDA/AMPA ratio: 1.5), as also shown by (Smeal et al., 2008), although other authors (Kreitzer and Malenka, 2007; Ding et al., 2008) found different ratios, which could be due to recruitment of non-cortical fibers by the positioning of the stimulating electrode within the striatum or external capsule, or age-dependent differences. Our data thus suggest that of all excitatory synapses on MSNs, those from the CL are likely to be the most effective in the depolarizing control of MSN firing, those from Pf are likely to be responsible for most of the Ca2+ influx, whereas those from the cortex are likely to contribute to both aspects.

Plasticity at thalamostriatal synapses

Synaptic plasticity at excitatory synapses in the striatum is complicated; both LTD and LTP have been reported and are variously dependent on the presence of neuromodulators, frequency of stimulation and/or pairing with postsynaptic action potentials (Mahon et al., 2004; Calabresi et al., 2007; Surmeier et al., 2007; Kreitzer and Malenka, 2008). We found that pairing optically evoked stimulation of thalamic synapses together with postsynaptic action potentials (with a slight delay) leads to robust LTD at Pf, but not CL, synapses. This plasticity is blocked if the calcium chelator, EGTA, or the NMDA receptor antagonist, MK801, is included in the intracellular solution suggesting that the plasticity is post-synaptically mediated and results from calcium influx through NMDA receptors. Previous studies have shown an involvement of dopamine (Pawlak and Kerr, 2008), endocannabiniods (Kreitzer and Malenka, 2007) and mediators released from striatal interneurons (Pakhotin and Bracci, 2007) in plasticity of cortical synapses. Although we did not test for these possibilities in the present study, it may well be the case that certain forms of plasticity at Pf (and potentially CL) synapses are also dependent on neuromodulators. It should also be noted that the calcium inflow might also modulate the induction of plasticity at other synapses on MSNs (Popescu et al., 2007) or, depending on location of the synapse (spine vs. dendrite), may initiate local dendritic spikes facilitating cooperative plasticity (Golding et al., 2002).

The thalamostriatal pathway is markedly heterogeneous, arising not only from CL and Pf but many other sub-nuclei of the thalamus (Groenewegen and Berendse, 1994; Smith et al., 2004). The present study shows that it is possible to use small volume, localized AAV-ChR2 injections to label and selectively activate small subnuclei of brain structures and demonstrates that the principal excitatory synapses from CL, Pf and cortex on MSNs exhibit markedly different properties, suggesting a differential contribution to information processing in the striatum. Finally, our findings of the properties of CL and Pf synapses are very reminiscent of the proposal that thalamic neurons fulfill the role of 'drivers' or 'modulators' (Sherman and Guillery, 1998). Thus CL synapses may act as drivers of MSN spiking activity whereas Pf synapses may subserve the role of modulators of the activity of MSNs. (Lee and Sherman, 2011).

Acknowledgements and funding

This research was supported by the European Community (FP7: HEALTH-F2-2008-201716) and the Medical Research Council, U.K. (grant U138164490).

Conflicts of Interests

The authors report no conflicts of interests.

Data Access

All data is published and accessible (Irino & Tada 2009)

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Figure legends

Figure 1: Localized transfection of thalamic neurons in the central lateral or parafascicular thalamic nucleus

(A) AAV particles containing the double floxed sequence for the light activatable channel channelrhodopsin-2 and YFP were injected into the intralaminar nuclei. Infected neurons expressing cre recombinase under the CAMKII promoter enables expression of ChR2-YFP (B) Diagram of a sagittal section of injected mouse brains. Small injections were made in either the rostral portion (mostly CL, green) or caudal portion (mostly Pf, red) of the intralaminar nuclei. (C & D) Coronal sections (rostral at top) of the right hemisphere of injected CAMKII-cre mice, which were injected in either the rostral CL (C) or caudal Pf (D) thalamic nucleus. Infected neurons are labeled with YFP (green). Sections have been stained with Nissl-Cy5 to facilitate anatomical characterization (blue). DG: dentate gyrus, LV: lateral ventricle, CA3: CA3 field of the hippocampus: fr: fasciculus retroflexus, mt: mammillothalamic tract, Mhb: medial habenula, Lhb: lateral habenula. Sourced from some citation.

Figure 2: The majority of MSNs receive both thalamic and cortical input

(A) Diagram of experimental setup for optical activation of either CL (i) or Pf (ii) thalamic afferents with stimulating electrode in external capsule for electrical activation of cortical afferents together with whole-cell patch-clamp recordings of MSNs. (B) Coronal section showing IRDIC visualization of section with superimposed fluorescence of same section showing the YFP-expressing thalamic fibers originating from the rostral intralaminar nuclei. (C) Confocal image of an MSN recorded and labeled with biocytin that was PPE-immunonegative and thus classified as a direct pathway MSN. (D) Confocal image of an MSN recorded and labeled with biocytin that was PPE-immunopositive and classified as a indirect pathway MSN (E) Response of the PPE-immunonegative MSN to electrical cortical and thalamic input arriving from CL (F) Example response of a PPE-immunopositive MSN to cortical and thalamic input arriving from from Pf.

Figure 3: Dynamic properties of CL, Pf and cortical synapses on MSNs

(A) Paired-pulse ratio of CL, Pf and cortical synapses on MSNs. Note that each input has a distinct PPR. On average the input from CL is significantly facilitating, Pf is depressing whereas input from the cortex is neutral (grey and black). Diamond symbols in the histogram indicate PPR of individual neurons (mean +- SD?) (B) Short-term dynamics at 5 Hz, 10 Hz and 20 Hz at CL (i), Pf (ii) and cortical (iii) synapses onto MSNs. Note the lack of short term dynamics using 5 Hz stimulation. Note the facilitating response at CL synapses at 10 Hz and 20 Hz (cross symbol). Note the quick recovery of the cortical input onto MSNs (asterisk symbol).

Figure 4: NMDA / AMPA receptor ratio at CL, Pf and cortical synapses on MSNs Postsynaptic ex