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• W2170686906 abstract "The excitatory afferents to the striatum from the cortex and thalamus are critical in the expression of basal ganglia function. Thalamostriatal afferents are markedly heterogeneous and arise in different subnuclei of the intralaminar thalamus. We used an optogenetic approach, to isolate and selectively activate thalamostriatal afferents arising in the central lateral or parafascicular thalamic nuclei, and to study the properties of their synapses with principal striatal neurons, the medium-spiny neurons. Thalamostriatal synapses differ in many aspects and our data suggest that inputs from the central lateral nucleus are efficient drivers of medium-spiny neuron action potential firing, whereas inputs from the parafascicular nucleus are likely to be modulatory. These results suggest distinct roles for thalamostriatal inputs from different subnuclei of the thalamus and will help us understand how the striatal circuit operates in health and disease. 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, as well as their ability to activate the main neurons of the striatum, the medium spiny neurons (MSNs). As the thalamostriatal projection is heterogeneous, we set out to isolate and study the thalamic 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 regions of the intralaminar thalamic nuclei (i.e. the central lateral or parafascicular nucleus). This enabled optical activation of specific thalamic afferents combined with whole-cell, patch-clamp recordings of MSNs and electrical stimulation of cortical afferents, in adult mice. We found that thalamostriatal synapses differ significantly in their peak amplitude responses, short-term dynamics and expression of ionotropic glutamate receptor subtypes. Our results suggest that central lateral synapses are most efficient in driving MSNs to depolarization, particularly those of the direct pathway, 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 Ca2+-dependent processes. Indeed, pairing parafascicular, but not central lateral, presynaptic stimulation with action potentials in MSNs, leads to NMDA receptor- and Ca2+-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. 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 & Knowlton, 2006). The striatum is the main input nucleus of the basal ganglia receiving excitatory afferents from the cortex and the thalamus (Kemp & Powell, 1971; Buchwald et al. 1973; Smith et al. 2004). 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 & Groenewegen, 1990; Castle et al. 2005; Smith et al. 2009). Both thalamic projections make direct synaptic contacts with the main classes of medium spiny neuron (Xu et al. 1991; Sadikot et al. 1992; Lacey et al. 2007). The Pf afferents, moreover, make contact with several types of striatal interneuron (Lapper & Bolam, 1992; Rudkin & Sadikot, 1999; Sidibé & Smith, 1999). Behavioural studies, mainly focused on Pf, have suggested thalamic intralaminar involvement in a variety of processes (Van der Werf et al. 2002; Smith et al. 2004; Minamimoto et al. 2005), including conveying behaviourally significant sensory signals (Matsumoto et al. 2001) and attentional values (Kinomura et al. 1996) to the striatum. Although the thalamostriatal and corticostriatal pathways give rise to similar numbers of synapses on MSNs (Lacey et al. 2005; Fujiyama et al. 2006; Raju et al. 2006; Moss & 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 can be overcome, to some extent, when studying the projection as a single entity, by careful placement of stimulating electrodes and/or careful selection of the slicing plane in vitro (Ding et al. 2008; Smeal et al. 2008). However, electrical stimulation cannot isolate different subnuclei of the thalamostriatal system. It is clear that these 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. 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 subnuclei of the intralaminar thalamus have different functional properties. To address this we set out to isolate and selectively activate the thalamostriatal projection originating in either the rostral or caudal portion of the intralaminar nuclei using targeted viral delivery of channelrhodopsin-2 (ChR2) (Boyden et al. 2005). Whole-cell patch-clamp recordings of neurochemically identified MSNs together 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 synapses, in the adult mouse striatum. We conclude that thalamostriatal synapses with striatal principal neurons show a hitherto unidentified heterogeneity which suggests they subserve different roles in striatal computation. All experiments were carried out on transgenic mouse lines which were bred and housed in accordance with the Animals (Scientific Procedures) Act 1986. 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. Adeno-associated virus serotype 2 (AAV2) carrying fusion genes for ChR2 or ChETA and YFP were injected into thalamus or cortex of CAMKII-cre mice between postnatal day 14 and 21. CAMKII is expressed in excitatory (glutamatergic) neurons and these transgenic mice generate cre recombinase in excitatory neurons, including corticostriatal and thalamostriatal neurons. 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. Typical coordinates from Bregma for cortical injections were lateral, 0.7 mm; posterior, 0.1 mm; and 0.3 mm ventral to the surface of brain. Briefly, CAMKII-cre mice were anaesthetized with isoflurane (2–4% in oxygen) and given analgesics (∼20 μl of 1:10 dilution of 0.3 mg ml−1 buprenorphine solution; Vetergesic; RHS, Slough, UK). A small craniotomy was made over the injection location. Viral particles were delivered through a 33-gauge needle using a Hamilton Microliter syringe placed in a Micro Syringe Pump Controller (Micro4, WPI) at a rate of 0.1 μl min−1. Injection volumes were 300 nl. Following a 3 min wait after injection the needle was retracted by 0.2 mm and, after another 3 min wait, slowly and fully retracted and the skin sutured. Topical analgesics were applied to the skin around the suture (Marcain) and subcutaneous injection of 5% glucose-saline solution given. Viral DNA included the double-floxed sequence for ChR2(H134R)-EYFP or ChR2(E123T-H134R)-EYFP driven by the elongation factor 1 promoter. AAV2 particles were produced at the University of North Carolina Gene Therapy Center Virus Vector Core. Typical titers were ∼1012 IU ml−1. After allowing 6–12 weeks for ChR2 or ChETA expression, acute striatal slices were prepared. Initial experiments to determine the correct injection coordinates and injection procedure consisted of injections of red latex beads (Lumafluor) together with immunostaining for cerebellin-1. Oblique coronal striatal slices (300–400 μm) were prepared from 2 to 4-month-old injected CAMKII-cre mice. Mice were anaesthetized with isoflurane (2–4% in oxygen) and decapitated. Slices were prepared in artificial cerebrospinal fluid (ACSF) containing (in mm): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 5 MgCl2, 2.5 CaCl2, 24 NaHCO3, and 10 glucose, pH 7.2–7.4, bubbled with carbogen gas (95% O2–5% CO2). Slices were immediately transferred to a storage chamber containing ACSF (in mm): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 1.5 MgCl2, 2.5 CaCl2, 24 NaHCO3, and 10 glucose, pH 7.2–7.4, bubbled with carbogen gas, at 37°C for 30 min and subsequently maintained at room temperature until used for 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−1 biocytin (pH 7.2–7.3; osmolarity, 290–300 mosmol l−1); and for whole-cell voltage-clamp (in mm): 120 caesium gluconate, 40 HEPES, 4 NaCl, 2 ATP-Mg, 0.3 Na-GTP, 0.2 QX-314 and 4 mg ml−1 biocytin (pH 7.2–7.3; osmolarity, 290–300 mosmol l−1). In a subset of synaptic 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). All recordings were performed at 32°C. 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 μA and 0.1 ms duration. Optical stimulation of thalamostriatal or corticostriatal 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 steady-state light power at the tissue was measured using a PMD100D power metre and photodiode sensor (Thor Labs). The spot size corresponded to the area of the slice visualized using a 40×/0.8NA water immersion objective, i.e. approximately 220 μm diameter. In some experiments we attempted to stimulate single thalamic afferents with a 473 nm diode laser (max. 100 mW output; UGA-40; Rapp OptoElectronic) coupled to the microscope with a single mode optic fibre (S405; Thorlab; 10 μm diameter spot; 3 ms duration pulses). Activation of excitatory afferents was performed in the presence of blockers of inhibitory GABAergic transmission including the GABAA-receptor antagonist SR95331 (10 μm) and GABAB-receptor antagonist CGP52432 (2 μm). Fibres were activated every 10 s 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 s for up to 20 times. Trains of pulses consisted of nine pulses at 5, 10 and 20 Hz, with the latter two followed by a recovery pulse 500 ms later, and was repeated every 30 s for up to 5 times. Combined AMPA and NMDA receptor-mediated currents were recorded from MSNs held at +40 mV. AMPA receptor-mediated currents were recorded after 5 min wash-in of d-AP5 (50 μm). Both consisted of at least 20 evoked responses at 0.1 Hz. The 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 time difference. 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. Data was analysed offline using custom written procedures in Igor Pro (Wavemetrics). EPSCs or EPSPs were defined 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 EPSC (S2) with the average slope of the first EPSC (S1). In plasticity experiments the paired-pulse ratio was determined prior to the induction protocol and at the end of the recording. Slopes of individual EPSCs were determined between 20% and 80% of maximum EPSC amplitude. Trains were analysed by taking the amplitude of each EPSC and dividing this by the amplitude of the first EPSC. The analysis of EPSC and EPSP basic properties (peak amplitude, duration, rise time (between 20% and 80% from peak) and decay time) was performed on individual synaptic responses. EPSCs obtained using minimal stimulation were defined as downward deflections of more than 3 standard deviations (SD) above baseline. 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 consecutive EPSPs was plotted. Plastic changes at synapses were assessed by comparing the average amplitude of recorded EPSPs during the last five minutes post induction with the baseline. The input resistance was constantly monitored by applying short –200 pA current steps to the patch pipette. If the input resistance changed more than 20% the recording was discarded. 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 4°C. After washes the slices were embedded in 5% agar and resectioned at 50 μm 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-100 (PBS-Tx) overnight at 4°C. YFP expression in thalamic fibres was visualized by incubating sections in 1:1000 chicken anti-GFP (Aves Labs) in PBS-Tx and 1% NDS overnight at 4°C 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 recorded MSN, the sections were heated at 80°C 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 4°C 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 overnight in 4% paraformaldehyde and 15% saturated picric acid in 0.1 m phosphate buffer (PB; pH 7.4) at 4°C. After washes, the brain blocks were resectioned at 50 μm on a vibrating microtome (VT1000S; Leica Microsystems). The sections were incubated in 20% NDS (Vector Laboratories) for 45 min at room temperature. A subset of sections was heated at 80°C in 10 mm sodium citrate (pH 6.0) for approximately 30 min prior to incubation with 1:2500 rabbit-anti-cerebellin-1 (Wei et al. 2007) In all sections YFP was visualised by immunolabelling using 1:1000 chicken-anti-GFP (Aves Labs) in PBS-Tx and 1% NDS overnight at 4°C followed by 1:500 donkey-anti-chicken-488 fluophore (Jackson Immunoresearch Laboratories), and in a subset of sections, 1:500 donkey-anti-rabbit-Cy3 fluophore (Jackson Immunoresearch Laboratories), 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, Göttingen, Germany) confocal microscope using ZEN and Axiovision software (Zeiss, Göttingen, Germany). The software's default settings for fluorophores were used for beamsplitters and ranges of emissions sampled. All data are presented as means ± SEM, except where stated. Student's t test, Wilcoxon's signed-rank test and the Mann–Whitney U test were performed using SPSS 17.0 (*P < 0.05, **P < 0.01). All drugs were obtained from Tocris Biosciences (Bristol, UK) and Sigma-Aldrich (Poole, Dorset, UK). The main thalamic input to the striatum comes from the rostral (central lateral) and caudal (parafascicular) region 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 in either the rostral or caudal portion of the intralaminar nuclei of CAMKII-cre mice (Fig. 1A and B). These transgenic mice generate cre recombinase in all CAMKII-expressing neurons and this approach allows expression of the light activatable channel only in the excitatory thalamic neurons around the site of injection. The AAV used is replication deficient and AAV serotype 2 was chosen, over e.g. AAV serotype 5, as it has been shown to enable focal neuronal transfection (Burger et al. 2004). After an initial iterative process, consisting of injections of latex beads in different locations (Supplemental Fig. S1A) and assessing the spread of the AAV transfection using different injection volumes in combination with immunolabelling of the thalamic nuclei (Wei et al. 2007) (Supplemental Fig. S1B), we were able to selectively inject either nucleus with little overlap, as assessed by the localization of YFP-expressing neurons (Fig. 1C and D). This suggests that transfection was predominantly localized to the CL or Pf subnuclei of the intralaminar thalamus. The transfected thalamic neurons for both nuclei covered an anterioposterior distance of approximately ∼750 μm (CL; 764 ± 85 μm and Pf; 706 ± 66 μm; n= 7 and 8), consistent with previous reports (Cardin et al. 2009). Although we found little to no transfection of cortical neurons we did find, particularly for rostral injections, occasional transfection of hippocampal neurons, but as the hippocampal projection is predominantly to ventral striatum we did not see this as a major issue. Localized transfection of 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 thalamic intralaminar nuclei. Infected neurons expressing cre-recombinase under the CAMKII promoter will start expressing ChR2-YFP. B, diagram of a sagittal section of the mouse brain indicating where small injections were made i.e. the rostral portion (mostly CL, green) and caudal portion (mostly Pf, red) of the thalamic intralaminar nuclei. C and D, coronal sections (rostral to caudal from top to bottom) of the right hemisphere of injected CAMKII-cre mice, which were injected in either the rostral CL (C) or caudal Pf (D) thalamic nucleus. Note that the sections shown for the CL injection start more rostral than those for the PF injection. Infected neurons express 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. 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 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 fibres (Fig. 2B). We observed a difference between the CL and Pf injected animals. Whereas the entire dorsal striatum had strong expression of YFP-positive fibres in CL-injected animals (Supplemental Fig. S2A and B), the distribution was more patchy in Pf-injected animals (Supplemental Fig. S2C and D). This is likely to reflect the different patterns of arbourization of the axons of CL and Pf neurons in the striatum (Lacey et al. 2007), but could also be the result of partial transfection of the Pf nucleus due to its larger size (see Fig. 1C and D). We also observed YFP-positive fibres in the cortex as a consequence of the expression of ChR2-YFP in the thalamic axonal projections to the cortex (Krettek & Price, 1977; Herkenham, 1979; Groenewegen, 1988). Whole-cell, voltage-clamp and current-clamp recordings of dorsal 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 (Fig. 2A). All the experiments were performed in the presence of the GABAA-receptor antagonist SR95331 (10 μm) and the GABAB-receptor antagonist CGP52432 (2 μm) and at a recording temperature of 32°C. The subtype of MSN was confirmed post hoc by immunolabelling for preproenkephalin (PPE) which is selectively expressed by indirect pathway MSNs (Gerfen et al. 1990) (Fig. 2C and 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 observed in both PPE– and PPE+ MSNs (Fig. 2E and F). The majority of MSNs receive both thalamic and cortical input A, diagram of the experimental setup for optical activation of either CL (a) or Pf (b) thalamic afferents and electrical activation of cortical afferents together with whole-cell patch-clamp recordings of MSNs. B, infrared differential interference contrast image of section with inset depicting fluorescence image of same region showing the YFP-expressing thalamic fibres originating from CL. C, confocal image of an MSN recorded and labelled with biocytin that was PPE-immunonegative and thus classified as a direct pathway MSN. D, confocal image of an MSN recorded and labelled with biocytin that was PPE-immunopositive and classified as an indirect pathway MSN. E, response of the PPE-immunonegative MSN to electrical stimulation of the cortex and optical stimulation thalamic afferents from CL. F, example response of a PPE-immunopositive MSN to electrical stimulation of the cortex and optical stimulation thalamic afferents from from Pf. 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 ∼3.5 mW, corresponding to ∼0.1 mW at the tissue, 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 (CL: 18.1 ± 3.5 pA and Pf: 9.1 ± 1.3 pA; P < 0.05; independent samples t test; n= 19 and n= 12) and Pf EPSCs slightly longer in duration (CL: 27.3 ± 2.3 ms and Pf: 35.0 ± 3.8 ms; P < 0.05; independent samples t test; n= 19 and n= 12). In all subsequent experiments we used higher stimulation strength to elicit approximately half maximum amplitude responses. The average amplitude of responses following CL axon stimulation was 66.3 ± 13.5 pA (stimulation strength: 45 mW at LED/0.4 mW at tissue; n= 16), whereas those following Pf axon stimulation were significantly smaller at 30.7 ± 4.1 pA (stimulation strength: 98 mW at LED/0.9 mW at tissue; P < 0.05; independent samples t test; n= 20). 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 PPE immunolabelling. 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: CL, 104.3 ± 33.8 pA vs. Pf, 28.7 ± 9.3 pA; P < 0.05; independent samples t test; both n= 5, and indirect pathway: CL, 40.8 ± 8.6 pA vs. Pf, 28.7 ± 8.6 pA; n= 7 and n= 9; Table 1). Furthermore, the analysis revealed that the response to CL axon stimulation was significantly larger in amplitude on direct pathway neurons (P < 0.05; independent samples t test; Table 1). Recordings performed in whole-cell current-clamp mode revealed a similar picture with an average amplitude of responses following CL axon stimulation of 4.0 ± 0.3 mV (stimulation strength: 52 mW at LED/0.5 mW at tissue; n= 40), whereas those following Pf axon stimulation were significantly smaller at 0.8 ± 0.1 mV (stimulation strength: 70 mW at LED/0.6 mW at tissue; P < 0.05; independent samples t test; n= 25). The response to CL axon stimulation was also consistently larger in amplitude than the response to Pf axon stimulation for both direct and indirect pathway MSNs (both P < 0.05; independent samples t test; n= 12 and n= 6, and n= 10 and n= 6; Table 1). Lastly, the analysis revealed that the response to CL axon stimulation was again significantly larger in amplitude on direct pathway neurons (P < 0.05; independent samples t test; Table 1). The responses obtained with LED stimulation are likely to be a consequence of the stimulation of multiple axons rendering the interpretation of different peak amplitudes of the responses uncertain. In order to facilitate the isolation of single axons and obtain a measure of the amplitude of an unitary response the experiment was repeated in whole-cell, voltage-clamp mode in which afferents were activated using 473 nm laser stimulation (100 mW maximum output) with a 10 μm spot diameter (Supplemental Fig. S3A). Initially, the stimulation intensity was moderate, 50% of maximum output at the laser, corresponding to 0.13 mW at the tissue, to aid in the detection of connections between fibres and MSNs. When an EPSC was observed, the stimulation intensity was reduced until only failures could be observed. The stimulation intensity was then incrementally changed with 5% step increases in laser output power until all stimulations led to a successful EPSC as measured from the postsynaptic neuron. Responses were accepted as unitary if the amplitude remained constant over a range where failures dropped. Averaging the responses within this range (100% failures – 0% failures) revealed that EPSCs for the CL synapses were significantly larger than for the Pf synapses (CL: 38 ± 10.3 pA and Pf: 14.7 ± 1.6 pA; P < 0.05; independent samples t test; n= 25 and n= 33; Supplemental Fig. S3B and C). Similar amplitude responses could also be seen occurring spontaneously during the recordings. These results suggest, first, that thalamic inputs from CL produce a larger depolarization of both direct and indirect pathway MSNs than Pf inputs. Secondly, CL inputs to direct pathway MSNs produce a stronger response than those to indirect pathway 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 (mean ratio of S2/S1: 1.11 ± 0.05; P < 0.05; one sample t test; n= 56), whereas those from Pf are depressing (mean ratio of S2/S1: 0.80 ± 0.08; P < 0.05; one sample t test; n= 22). 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" @default.
• W2170686906 created "2016-06-24" @default.
• W2170686906 creator A5021880324 @default.
• W2170686906 creator A5023155664 @default.
• W2170686906 creator A5039227814 @default.
• W2170686906 creator A5050827322 @default.
• W2170686906 creator A5070952959 @default.
• W2170686906 date "2013-01-01" @default.
• W2170686906 modified "2023-10-16" @default.
• W2170686906 title "Heterogeneous properties of central lateral and parafascicular thalamic synapses in the striatum" @default.
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