Distinct cellular properties of identified ... - Wiley Online Library

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J Physiol 589.15 (2011) pp 3775–3787

Distinct cellular properties of identified dopaminergic and GABAergic neurons in the mouse ventral tegmental area Billy Chieng, Yael Azriel, Sarasa Mohammadi and MacDonald J. Christie

The Journal of Physiology

Brain and Mind Research Institute, University of Sydney, NSW 2006, Australia

Non-technical summary The lower mid-brain of rodent is home to addiction and hedonism of substances of abuse. Due to overlapping physiological properties among different types of nerve cell within this brain region, it has been difficult to selectively study the physiology of a single type of nerve cell. Here, using gene technology together with selective antibody detection, we have exclusively identified the two dominant populations of nerve cells and found dopamine-containing cells five times more abundant. We found clear non-overlapping cellular characteristics between the two types that are unequivocal predictors for selection of nerve cells, whilst other previously employed cellular criteria were found to be less useful. Abstract The midbrain ventral tegmental area (VTA) contains neurons largely with either a dopaminergic (DAergic) or GABAergic phenotype. Physiological and pharmacological properties of DAergic neurons have been determined using tyrosine hydroxylase (TH) immunohistochemistry but many properties overlap with non-DAergic neurons presumed to be GABAergic. This study examined properties of GABAergic neurons, non-GABAergic neurons and TH-immunopositive neurons in VTA of GAD67-GFP knock-in mice. Ninety-eight per cent of VTA neurons were either GAD-GFP or TH positive, with the latter being five times more abundant. During cell-attached patch-clamp recordings, GAD-GFP neurons fired brief action potentials that could be completely distinguished from those of non-GFP neurons. Pharmacologically, the μ-opioid agonist DAMGO inhibited firing of action potentials in 92% of GAD-GFP neurons but had no effect in non-GFP neurons. By contrast, dopamine invariably inhibited action potentials in non-GFP neurons but only did so in 8% of GAD-GFP neurons. During whole-cell recordings, the narrower width of action potential in GAD-GFP neurons was also evident but there was considerable overlap with non-GFP neurons. GAD-GFP neurons invariably failed to exhibit the potassium-mediated slow depolarizing potential during injection of positive current that was present in all non-GFP neurons. Under voltage-clamp the cationic current, I h , was found in both types of neurons with considerable overlap in both amplitude and kinetics. These distinct cellular properties may thus be used to confidently discriminate GABAergic and DAergic neurons in VTA during in vitro electrophysiological recordings. (Received 18 April 2011; accepted after revision 30 May 2011; first published online 6 June 2011) Corresponding author B. Chieng: Brain and Mind Research Institute, University of Sydney, Level 6, Building F, 94 Mallett Street, M02F, Camperdown, NSW 2050, Australia. Email: [email protected] Abbreviations 3n, oculomotor nerve; D2, dopamine receptor type 2; DA, dopamine; DAMGO, [D-Ala2 , N -Me-Phe4 , Gly5 -ol]-enkephalin; fr, fasciculus retroflexus; GAD, glutamic acid decarboxylase; GIRK, G-protein-coupled inwardly-rectifying potassium channel; I h , hyperpolarization-activated cation current; IP, interpeduncular nucleus; ml, medial lemniscus; IPF, interpeduncular fossa; MG, midline group; MT, medial terminal nucleus of accessory optic tract; SNc, substantia nigra pars compacta; TH, tyrosine hydroxylase; VGLUT2, vesicular glutamate transporter type 2; VTA, ventral tegmental area.

C 2011 The Authors. Journal compilation C 2011 The Physiological Society

DOI: 10.1113/jphysiol.2011.210807

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B. Chieng and others

Introduction The majority of neurons in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) are either dopaminergic (DAergic) or GABAergic (Lacey et al. 1989; Yung et al. 1991; Johnson & North, 1992b), with only a few per cent expressing markers for neither neurotransmitter (they are glutamatergic) (Nair-Roberts et al. 2008). Early cellular physiological studies in vitro classified neurons in VTA and SNc as principal (DAergic) or secondary (presumed GABAergic) on the basis of distinct physiological and pharmacological properties (Grace & Onn, 1989; Lacey et al. 1989; Johnson & North, 1992a,b) combined with post hoc tyrosine hydroxylase (TH) immunohistochemistry (Grace & Onn, 1989; Yung et al. 1991; Johnson & North, 1992b). Briefly, DAergic neurons were considered to exhibit slow regular firing of broad action potentials, a prominent hyperpolarization-activated cation current (I h ) and were hyperpolarized by DA via D2 receptors but not by μ-opioid agonists. By contrast, non-DAergic (presumed GABAergic) neurons often spontaneously fired high frequency, short duration action potentials, did not exhibit I h and were hyperpolarized by μ-opioid agonists but not DA. Whilst physiological and pharmacological properties can effectively distinguish many DAergic from non-DAergic neurons in SNc (e.g., Lacey et al. 1989), this can be confounded in VTA because there is considerable overlap of properties (Ford et al. 2006; Margolis et al. 2006, 2008; Lammel et al. 2008). Therefore, definition of neurotransmitter phenotype of VTA neurons on the basis of action potential properties, I h and responses to DA can be inaccurate. Using post hoc TH immunohistochemistry in rat, Margolis et al. (2006) reported that action potential duration and frequency, the presence of I h or actions of DA are variable and do not accurately predict whether or not a neuron is DAergic. However, complete absence of I h did reliably predict that a neuron was not dopaminergic (TH negative). Ford et al. (2006) also identified a large range of expression of I h in mouse VTA DAergic neurons but suggested that action potential duration during cell-attached recordings could reliably discriminate DAergic (TH positive) from non-DAergic neurons. Lammel et al. (2008) also identified a subpopulation of mouse VTA DAergic neurons with high action potential activity but do not exhibit GIRK coupled DA D2 receptors. There is also strong evidence for subpopulation differences amongst DAergic VTA neurons based on projections to different targets (Ford et al. 2006; Lammel et al. 2008; Margolis et al. 2008) and cellular morphology (Sarti et al. 2007). For example, although VTA DAergic neurons that are localized adjacent to SNc exhibit a prominent I h , those located more medially express a much smaller I h (Ford et al. 2006; Sarti et al. 2007). Furthermore,

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many medial DAergic neurons that exhibit little I h and brief action potentials have been reported to project differentially to distinct target structures including nucleus accumbens, striatum, amygdala and prefrontal cortex (Ford et al. 2006; Lammel et al. 2008). Given that many presumed non-DAergic neurons in VTA also express I h (Jones & Kauer, 1999; Margolis et al. 2006), classifications based on the appearance I h alone are not adequate to confidently distinguish DAergic and GABAergic neurons. Potential false negative results from post hoc staining of TH after quantifying physiological properties may also have hindered unequivocal classification of non-DAergic neurons (Zhang et al. 2010). GABAergic neurons in VTA are often assumed to fire high frequency, brief duration action potentials and exhibit little or no I h (Grace & Onn, 1989; Johnson & North, 1992b). Whilst the proportions of non-DAergic neurons reported in such studies generally concurs with detailed immunohistochemical studies of GABAergic markers (Olson & Nestler, 2007; Nair-Roberts et al. 2008), the GABAergic phenotype has been confirmed using histochemical methods only in a few cases (Steffensen et al. 1998). Subgroups of presumed VTA GABAergic neurons have also been identified based on differences in spontaneous action potential frequency (e.g. Korotkova et al. 2004). The present study used enhanced green fluorescent protein knock-in mice targeting the GAD67 locus (GAD-GFP mice) to unambiguously label GABAergic neurons in VTA (Tamamaki et al. 2003), together with TH immunohistochemistry. We found that GAD-GFP neurons were less abundant but well mixed among TH positive neurons in VTA. Mapping of co-staining together with NeuN confirmed that GAD-GFP and TH positive neurons are mutually exclusive and comprise the majority of VTA neurons, with a very small subpopulation stained neither by TH nor GFP antibodies. In patch clamp recordings from brain slices, it was observed that GAD-GFP neurons had a brief action potential duration that did not overlap with non-GFP neurons, particularly during cell-attached patch clamp recordings. Pharmacological responses to DA and a μ-opioid agonist also distinguished most DAergic and GABAergic neurons during cell-attached recordings. During whole-cell recordings, evoking action potentials by depolarizing current injection invariably produced delayed action potential initiation in non-GFP neurons but not in GAD-GFP cells. Other properties such as action potential frequency, duration in whole cell recording and presence of I h were less useful. Physiological properties of GAD-GFP and TH neurons in VTA can therefore be routinely and unequivocally distinguished during cell-attached patch-clamp recordings on the basis of duration of the action potential duration waveform and can be confirmed by pharmacological responses. C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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GABA and dopamine neurons in VTA

Methods Animals and ethical approval

GAD67 knock-in mice were generously provided by Professor T. Kaneko (Tamamaki et al. 2003). Briefly the GAD67 locus of the knockout was targeted by homologous recombination with enhanced-GFP cDNA. Only heterozygous animals were viable. Forty-seven male mice were used in the study. All animal experiments were approved by the University of Sydney and Royal North Shore Hospital/University of Technology Sydney Ethics Committees, which comply with National Health and Medical Research Council of Australia guidelines and NSW legislation. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Brain slice preparation and electrophysiological recording

Mice (4–8 weeks old) were killed under deep anaesthesia by isoflurane inhalation (4% in air) and decapitated, and the brain was removed. Horizontal brain slices (240 μm thick) containing VTA were cut using a vibrating tissue slicer (Leica VT1000S, Germany) in ice-cold oxygenated sucrose cutting solution containing (in mM): 241 sucrose, 28 NaHCO3 , 11 glucose, 1.4 NaH2 PO4 , 3.3 KCl, 0.2 CaCl2 , 7 MgCl2 . Slices were maintained at 33◦ C in a submerged chamber containing physiological saline equilibrated with 95% O2 and 5% CO2 . The slices were then transferred to a recording chamber and superfused continuously (1.5 ml min−1 ) with physiological saline of composition (in mM): 126 NaCl, 2.5 KCl, 1.4 NaH2 PO4 , 1.2 MgCl2 , 2.4 CaCl2 , 11 glucose and 25 NaHCO3 . All experiments were conducted on VTA neurons located in the DAergic process rich region medial to substantia nigra pars compacta. VTA neurons were visualized on an upright microscope (Olympus BX50WI) using infra-red Normarski or differential interference contrast Dodt tube optics. Cell-attached and whole-cell patch-clamp recordings were made using electrodes (2–5 M) containing (in mM): 115 KCH3 SO4 , 15 NaCl, 1 MgCl2 , 10 Hepes, 11 EGTA, 5 Mg-ATP, and 0.33 Na-GTP, pH 7.3, osmolarity 285–290 mosmol l−1 . Biocytin (0.1%) was routinely added to the internal solution for marking the recorded neurons. Whole-cell recordings were established immediately following data collection in cell-attached mode (usually less than 5 min). Series resistance (less than 20 M) was compensated by 80% and monitored periodically during experiments with an Axopatch 200A or Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA), connected to a Macintosh computer and ITC-18 (Instrutech, Long Island, NY, USA). Liquid junction potentials of C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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−10 mV were corrected. In cell-attached mode, action potentials were sampled at 10 kHz (low pass filter 5 kHz) and whole-cell currents were sampled at 5 kHz (low pass filter 2 kHz, Axograph X, Molecular Devices). Stock solutions of all drugs were diluted to working concentrations in the extracellular solution immediately before use and applied by continuous superfusion. Data from cell-attached and whole cell recordings were only included in analyses if action potential amplitudes were at least 55 mV after establishing whole-cell recording mode, to ensure that only highly viable neurons were included.

Immunohistochemistry

Male GAD-GFP mice (6–8 weeks old) were overdosed with pentobarbitone (300 mg kg−1 intraperitoneal) and transcardially perfused with a fixative containing 4% paraformaldehyde in phosphate buffer (PB). Brains were subsequently removed and postfixed overnight at 4◦ C. Cryoprotection was employed by placing brains in 30% sucrose solution for 2 days. Thin sections (30 μm) of mouse brain tissue containing the VTA were collected and stained as free floating sections. For immunohistochemistry, sections were incubated in 0.3% Triton X-100/PB for (1 h), 10% horse serum/PB (1 h), and then in primary antisera solution (1:1000 sheep anti-TH, Chemicon; 1:500 chicken anti-GFP, Aves; 0.1% bovine serum albumin; 1% horse serum and 0.1% Triton X-100/PB) for 3 days at 4◦ C. After briefly rinsing sections in PB, they were incubated in secondary antisera containing 1:200 Cy3-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), 1:200 Cy2-conjugated donkey anti-chicken IgY (Jackson), 0.1% bovine serum albumin; 1% horse serum and 0.1% Triton X-100/PB at room temperature (2 h). In another five mice, mouse anti-NeuN (1:1000, Chemicon) was added to the primary antisera incubation and Alexa 647-conjugated donkey anti-mouse IgG (1:200) in the secondary incubation to reveal the entire VTA neuronal population. Sections were rinsed and mounted onto gelatinized glass slides and coverslipped with an aqueous mounting medium (Vectashield, Vector Laboratories, Inc., Burlingame, CA, USA). For biocytin-filled neurons, immediately after brief physiological recording (5–10 min) to avoid loss of eGFP and TH immunoreactivity, brain slices were fixed overnight in 4% paraformaldehyde/0.16 M PB solution followed by placing them in 0.3% Triton X-100/PB for 3 days to permeabilize cell membrane. Slices were then placed in 10% horse serum/PB for 1 h before being incubated in primary sheep anti-TH (1:1000, Chemicon, Temecula, CA, USA) for 2 days at 4◦ C. The slices were rinsed in PB

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and then in a one-step incubation containing both Cy3-conjugated donkey anti-sheep secondary antibody (1:500, Jackson) and Alexa 647-conjugated Streptavidin (1:500, Invitrogen, Carlsbad, CA, USA) for 2 h. Stained slices were rinsed, mounted onto glass slides, dried and coverslipped with Vectashield mounting medium (Vector Laboratories).

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Chemicals used

Biocytin, DAMGO, dopamine and 4-aminopyridine were purchased from Sigma-Aldrich (St Louis, MO, USA). Results Immunohistochemistry and distribution of GAD-GFP and TH neurons

Microscopy

An Olympus microscope BX61WI with FluoView FV-300 version 4.3 confocal scanning system (lasers 488, 546 and 647) and software were used. Images were taken sequentially with different lasers with 10×, 40× and 60× oil immersion objectives and z-axis stacks were collected at 5, 2 and 0.5 μm, respectively. For counting GFP and TH positive neurons, the number of neurons was counted within a random 300 × 400 × 30 μm3 brain section by sequentially scoring positive cells through a 10-optical level image stack (per mouse). Images were taken with a 40× objective. The longest diameter of each neuron was measured from the confocal image stack using FluoView software. For assessing double and triple labelling of GFP, TH and NeuN, using FluoView, confocal images (60× objective, 200 × 200 × 30 μm3 images) from the three separate antibodies were overlaid while going through a 40-optical level image stack (per mouse). Projection images were exported as tiff files and locations of individual neurons were plotted in Adobe Illustrator with reference to standard horizontal atlas sections.

Data analysis

Coefficient of variation of action potential firing frequency was calculated by dividing the S.D. of inter-spike interval by the mean inter-spike interval. The spontaneous action potentials used for this analysis were sampled from a raw trace of 60 s duration. Action potential rise time was determined as the rise from 10 to 90% on the up-slope of action potential above threshold. The I h time-dependent rectification index was calculated as instantaneous current amplitude at the beginning of voltage step −130 mV subtracted from instantaneous current amplitude at the end of voltage step. Decay time constant (τ) of I h tail current was a measure of time taken for current reduction to 63% on the decay part of curve. The current–voltage raw trace used for measuring τ was one stepped to −130 mV. All pooled values are expressed as means ± SEM. Statistical tests between treatment groups were made using unpaired t tests. Differences among proportions was analysed using χ2 tests. Significance was accepted at P < 0.05.

As reported elsewhere in various mouse strains (Prasad & Richfield, 2008), the VTA contained a very high density of TH positive cell bodies and processes in GAD-GFP mice (Figs 1–3). Intense TH staining was evident in VTA – located medial to SNc and the medial optic tract (MT, Fig. 1). VTA neurons in co-stained sections were either TH or GAD-GFP positive, but not both (from 9 GAD-GFP mice). TH and GAD-GFP positive neurons were then plotted throughout the VTA. TH positive neurons were approximately 5 times more abundant than GAD-GFP cells (n = 5 mice, Fig. 2). GAD-GFP neurons were distributed quite evenly throughout VTA and mixed amongst TH neurons. Because a small proportion of VTA neurons have been reported to be non-TH, non-GAD, glutamatergic neurons (Yamaguchi et al. 2007), the entire neuronal population in VTA was stained with an anti-NeuN antibody whilst co-staining TH and GAD-GFP antibodies in the same sections. Nearly all NeuN positive neurons were co-stained for either TH or GFP (double-labelled TH + NeuN = 91 ± 4 neurons, 78%; double-labelled GFP + NeuN = 23 ± 3 neurons, 20%; and NeuN only non-TH non-GFP = 2 ± 1 neurons, 2%, n = 4 mice, Fig. 3, see Methods for cell counting). There was also no difference in neuron diameter (longest axis of soma) between TH and GAD-GFP neurons (length 17.5 ± 0.2 μm from 513 TH neurons, 17.4 ± 0.4 μm from 81 GAD-GFP neurons, P = 0.89, n = 4 mice, Fig. 1 and 2 bottom right, see Methods for cell measurement). Biocytin labelled neurons from electrophysiological recording

A total of 95 neurons (56 GAD-GFP and 39 non-GFP) from 38 mice were recorded in the present study (Fig. 4). From those, 43% (41 neurons total) of neurons were successfully recovered with co-staining of biocytin, GFP and TH in the same brain slices (Fig. 4A-C). A large range of cell diameters (12–50 μm) was observed in both the GAD-GFP and non-GFP neurons sampled during electrophysiological recording with GAD-GFP cells significantly smaller (longest soma diameter 18 ± 1 μm, n = 18 for GAD-GFP versus 26 ± 2 μm, n = 23 for non-GFP, P = 0.0005, Fig. 4D), but there was substantial overlap. C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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Cellular properties during cell-attached recordings unequivocally distinguish GAD-GFP from non-GFP neurons

Using expression of eGFP as an unequivocal index of GABAergic neurons, we found a number of physiological

characteristics that were clearly suitable for distinguishing GAD-GFP from non-GFP neurons. Using cell-attached patch-clamp recordings, spontaneous action potentials exhibited a distinctly shorter duration in GAD-GFP cells than in non-GFP cells (0.43 ± 0.02 ms, n = 18 GAD-GFP cells versus 1.49 ± 0.04 ms, n = 26 non-GFP

Figure 1. Distribution of TH positive and GAD-GFP positive neurons throughout the VTA A–E, rostro-caudal series of confocal images of coronal sections containing VTA (distance from bregma estimated from Franklin & Paxinos, 1997) in a GAD67-GFP mouse. VTA is marked by intense TH labelling (red). GAD-GFP neurons are green and TH neurons are red. MT is medial terminal nucleus of accessory optic tract, SNc is substantial nigra pars compacta, IP is interpeduncular nucleus, ml is medial lemniscus. F, a representative confocal image of VTA taken from a separate section in the same mouse (60× oil objective). Scale bar A–E, 300 μm and F, 30 μm. C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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cells, P < 0.0001, Fig. 5A and B). Indeed, there was no overlap of action potential duration between groups (Fig. 5B) and 99% confidence intervals of mean did not overlap (CI for GAD-GFP 0.37–0.49 ms versus CI

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for non-GFP 1.37–1.61 ms). Other parameters sampled during cell-attached recordings also differed significantly between groups, but overlapped somewhat, indicating that they may serve as useful secondary criteria to

Figure 2. Scored distribution of TH positive and GAD-GFP positive neurons in VTA A–E, distribution of GAD-GFP (green dots) and TH (red dots) neurons along the rostro-caudal axis of VTA that correspond to sections shown in Figure 1. F, summary of numbers of GAD-GFP and TH positive neurons identified per section along the rostrocaudal axis of VTA. Neurons were counted from confocal images 300 × 400 μm from single 30 μm sections sampled from each level in 4 mice. C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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distinguish GABAergic from non-GABAergic neurons. GAD-GFP neurons have a higher spontaneous action potential frequency than non-GAD neurons (3.6 ± 0.8 Hz, n = 27 GAD-GFP neurons versus 1.6 ± 0.2 Hz, n = 23 non-GFP neurons, P = 0.02, Fig. 5C), but the coefficient of variation indicated a significantly more irregular firing of spontaneous action potentials in GAD-GFP neurons (0.68 ± 0.17, n = 16 cells versus 0.18 ± 0.04, n = 19 non-GFP cells, P = 0.004, Fig. 5D). Like DAergic neurons in the SNc, action potential activity of those in VTA are inhibited by exogenously applied DA acting on D2 receptors (Johnson & North, 1992a,b; Cameron et al. 1997; Margolis et al. 2006). By contrast, presumed GABAergic interneurons are inhibited by μ-opioid agonists such as DAMGO (Johnson & North, 1992a,b; Cameron et al. 1997). The actions of DA and DAMGO on excitability of GAD-GFP and non-GFP neurons in VTA were therefore examined in

cell-attached patch-clamp mode. In GAD-GFP neurons, nearly all (11 out of 12) cells were inhibited by DAMGO (1 μM) but not dopamine (100 μM, 1 of 12 cells was inhibited, Fig. 5E). One GAD-GFP cell was inhibited by both DA and DAMGO whilst another GAD-GFP cell responded to neither. The converse was the case for non-GFP neurons. DA invariably inhibited spontaneous action potential activity whilst DAMGO has no effect in the same cells (n = 25, Fig. 5F).

Cellular properties during whole-cell recordings unequivocally distinguish GAD-GFP from non-GFP neurons

Action potentials were briefer in GAD-GFP than non-GFP neurons when examined in whole-cell recording mode. This was confirmed using both amplifiers

Figure 3. Most neurons in VTA are either TH positive or GAD-GFP positive Confocal images (45 adjacent 0.5 μm optical sections merged) of VTA neurons in a single brain section triple labelled with TH (A), GAD-GFP (B) and NeuN (C). D, a merged image of A–C. Arrows indicate neurons positive only for NeuN (non-TH and non-GFP positive). Scale bar is 50 μm. C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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(Axopatch 200A, 0.45 ± 0.02 ms, n = 23 GAD-GFP cells versus 1.28 ± 0.07 ms, n = 17 non-GFP cells, P < 0.0001; and Multiclamp 700B, 0.76 ± 0.07 ms, n = 4 GAD-GFP cells versus 1.66 ± 0.07 ms, n = 5 non-GFP cells, P < 0.0001, Fig. 6A and B). The 99% CI of mean was 0.41–0.58 ms for GAD-GFP versus 1.18–1.56 ms for non-GFP neurons. The rising phase of action potentials was also faster in GAD-GFP than non-GFP neurons (0.14 ± 0.01 ms, n = 27 cells versus 0.31 ± 0.02 ms, n = 22, respectively, P < 0.0001, Fig. 6C). Action potential duration in whole-cell recording mode was highly correlated with that determined during cell-attached recordings (Fig. 6D). Action potential amplitude above threshold was slightly larger in GAD-GFP than non-GFP neurons (72 ± 2 mV, n = 27 cells versus 65 ± 2 mV, n = 23, respectively, P = 0.009, not illustrated). Input conductance of GAD-GFP and non-GFP neurons did not differ significantly (from −60 mV to −80 mV, 2.2 ± 0.5 nS, n = 27 GAD-GFP cells versus 2.5 ± 0.4 nS, n = 23 non-GFP cells, P = 0.56).

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The presence or absence of a slow sub-threshold depolarizing potential preceding action potentials evoked by injection of a step depolarizing current from a hyperpolarized holding membrane potential (−90 mV) also completely discriminated GAD-GFP from non-GFP neurons. Unlike GAD-GFP neurons (n = 26), non-GFP VTA neurons (n = 25) invariably displayed a slow depolarizing potential (Fig. 6E). This slow sub-threshold depolarizing potential was completely abolished by potassium channel blockers such as 4-aminopyridine (4 mM, n = 4, Fig. 6E). The presence or absence of an I h current poorly distinguished GAD-GFP from non-GFP cells (Fig. 7). The presence or absence of I h was determined in voltage-clamp from the amplitude of the time-dependent inward rectification during a voltage step from −70 to −130 mV (b – a in Fig. 7Ad). Although most GAD-GFP neurons expressed little or no I h (Fig. 7A and B) and the amplitude was significantly less than in non-GFP cells (rectification index 0.018 ± 0.008 nA, n = 26 GAD-GFP cells versus

Figure 4. Post hoc identification of TH positive and GAD-GFP positive VTA neurons A, confocal images of two biocytin labelled neurons combined with post hoc immunohistochemistry. B, a TH positive neuron (arrow) was also labelled with biocytin in A. C, a GFP positive neuron (arrow) was also labelled with biocytin in A. D, diameters (longest axis) of biocytin-labelled GAD-GFP (n = 18) and TH positive (n = 25) neurons. E and F, anatomical locations of recorded VTA neurons plotted in horizontal brain slice orientation at two bregma levels. Green and red dots represent individual GFP and non-GFP neurons, respectively. MT is medial terminal nucleus, SN is substantial nigra, IPF is interpeduncular fossa, ml is medial lemniscus, fr is fasciculus retroflexus, MG is midline group and 3n is oculomotor nerve. C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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0.193 ± 0.049 nA, n = 23 non-GFP cells, P = 0.0005). A substantial proportion of non-GFP cells also failed to express I h (Fig. 7A and B). Analysis of I h tail-current decay time also indicated a smaller time constant (τ) in GAD-GFP neurons (121 ± 20 ms, n = 19 cells versus 397 ± 29 ms, n = 12 non-GFP cells, P < 0.0001, Fig. 7C), but again there was some overlap between groups and the measure may be confounded by outward tail currents, presumably due to recovery K+ current from inactivation (see Fig 7Ac). Discussion The present study used TH immunostaining in GAD-GFP knockin mice to determine whether DAergic and GABAergic neurons in mouse VTA can be distinguished reliably. The vast majority of neurons in mouse VTA

Figure 5. Physiological and pharmacological properties of VTA neurons recorded in cell-attached patch-clamp mode A, representative examples of action potentials of a GAD-GFP neuron (left) and non-GFP neuron (right). Duration of action potential was measured within the dotted line in horizontal axis. B, summary of action potential durations of GAD-GFP and non-GFP neurons. Circles represent neurons sampled with Axopatch 200A amplifier and triangles represent neurons recorded using a Multiclamp 700B. C, spontaneous action potential frequency for GAD-GFP and non-GFP neurons sampled for 60 s. D, coefficient of variation for spontaneous action potential frequency calculated by dividing SD of interspike interval by the mean of interspike interval. E, representative profile of most spontaneously active GAD-GFP neurons showing no response to superfusion of dopamine (DA, 100 μM) but complete, reversible inhibition of action potential activity by DAMGO (1 μM). F, representative profile of all spontaneously active non-GFP neurons showing no response to superfusion of DAMGO (1 μM) but complete, reversible inhibition of action potential activity by dopamine (100 μM). C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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were DAergic (defined by immunostaining for TH) or GABAergic (defined by expression of GFP; Tamamaki et al. 2003). Expression of GAD-GFP and TH immunostaining was mutually exclusive, with only about 2% of NeuN positive neurons expressing neither GAD nor TH. GABAergic neurons are less abundant and more uniformly distributed in mouse VTA than rat. Margolis et al. (2006) reported that 45% of neurons in VTA did not express TH, which corresponds well with the study of Nair-Roberts et al. (2008), who reported 35% were GABAergic with considerable variation among VTA subfields. Nair-Roberts et al. (2008) also reported that a very small proportion of VTA neurons (2–3%) express glutamatergic markers (VGLUT2). Although Yamaguchi et al. (2007) reported little overlap between TH and VGLUT2 expression, Kawano et al. (2006) found overlap in some subfields of VTA, consistent with findings

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that midbrain dopaminergic neurons projecting to the nucleus accumbens can synaptically co-release glutamate (Tecuapetla et al. 2010). Therefore it is possible that the small proportion of the non-TH, non-GAD-GFP neurons examined here represent the TH negative glutamatergic neurons previously reported in rat. It is not known whether any of the GAD-GFP negative neurons that were not confirmed to be TH positive after patch-clamp recording may have been glutamatergic because physiological properties of glutamatergic VTA neurons have not yet been determined (Kawano et al. 2006; Yamaguchi et al. 2007; Nair-Roberts et al. 2008). As previously reported in rat VTA (Margolis et al. 2006), mouse DAergic and GABAergic neurons could not be distinguished unequivocally using anatomical or morphological properties. Without careful consideration in methodology, even post hoc confirmation using TH from biocytin-labelled neurons during recording may

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still generate false negative data due to significant dialysis of cellular TH content after 15 min of whole-cell recording (Zhang et al. 2010). Thus, biocytin labellings of VTA neuron in the present study were restricted to 5–10 min under whole-cell recording. The mean diameters (measured along the longest axis) of DAergic neurons recovered and stained for TH or GFP after recording were significantly larger than GABAergic neurons, but there was considerable overlap as in rat VTA (Margolis et al. 2006). As in rat VTA (Olson & Nestler, 2007), GAD-GFP positive neurons were predominantly interspersed amongst TH neurons throughout the VTA. DAergic and GABAergic neurons could readily be distinguished on the basis of several easily acquired cellular physiological properties, particularly during cell-attached patch clamp recordings. Although many GAD-GFP neurons fired action potentials at quite high, variable frequencies compared with non-GFP neurons

Figure 6. Physiological properties of VTA neurons recorded in whole-cell patch-clamp mode A, action potentials of a GAD-GFP neuron (left) and a non-GFP neuron (right). Duration of action potential was measured within the dotted line in horizontal axis. B, summary of action potential durations of GAD-GFP and non-GFP neurons. Each circle represents a neuron sampled with Axopatch 200A amplifier and triangles represent neurons recorded using a Multiclamp 700B. C, action potential rise time (10–90%) for GAD-GFP and non-GFP neurons. D, correlation plot of action potential durations for GAD-GFP (open circles) and non-GFP (filled circles) neurons under cell-attached and whole-cell modes. The linear regression line is fitted. It has a slope of 0.96 and a correlation coefficient of 0.88. E, under whole-cell current clamp, action potentials from GAD-GFP (left) and non-GFP (middle) neurons were evoked by injection of a positive current. Each cell was held at −90 mV. Non-GFP VTA neurons typically exhibited a slow initial rising phase before triggering an action potential. Panel on the right shows that slow initial rising phase in the same non-GFP neuron was blocked by superfusion of 4-aminopyridine (4AP; 4 mM). C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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(e.g., Steffensen et al. 1998), others were quiescent, in agreement with micro-electrode recordings of presumed non-dopaminergic neurons in rats (Johnson & North, 1992b) but not mice (Ford et al. 2006). As suggested by Ungless et al. (2004), the most reliable single criterion was the duration of the action potential waveform during cell-attached recordings, with all spontaneously active neurons exhibiting a waveform duration (as defined in Fig. 5) of 0.43 ms versus a mean duration for non-GFP cells of 1.49 ms with well separated 99% confidence intervals. This finding is consistent with Ford et al. (2006), who reported no overlap in action potential duration between DAergic and non-DAergic neurons in mouse VTA, with a minimum duration of 1.2 ms for TH positive neurons and a maximum duration of 0.9 ms for presumed GABAergic neurons. The present results agree quite well with the action potential duration cut-off of 1.2 ms proposed by Ford et al. (2006) to discriminate mouse VTA DAergic from non-DAergic neurons. Spontaneously active GAD-GFP positive neurons were also inhibited by a μ-opioid agonist but not dopamine in all but

one case (92%), consistent with previous physiological classifications (Johnson and North, 1992a,b; Cameron et al. 1997; Ford et al. 2006) and electron microscopy (Garzon & Pickel, 2001). Thus combined use of action potential duration, μ-opioid and/or dopamine responsiveness during cell-attached recordings can unequivocally distinguish spontaneously active DAergic and GABAergic neurons in VTA. However, a considerable proportion (22%) of GAD-GFP positive neurons were not spontaneously active, as reported previously for rat TH negative neurons (Johnson & North, 1992b), precluding use of this cell-attached criterion for quiescent VTA neurons. This limitation could potentially be overcome in future by recording cell-attached properties in the presence of elevated extracellular K+ concentrations to drive action potentials in quiescent neurons. GAD-GFP neurons exhibited a greater range of spontaneous action potential frequency and fired action potentials more irregularly than non-GABAergic neurons (Grace & Onn, 1989; Johnson & North, 1992b).

Figure 7. I h currents in VTA neurons Aa–d, under whole-cell voltage clamp, neurons were held at −70 mV stepped to more negative potentials in 20 mV increments. Examples of GAD-GFP (a and b) and non-GFP neurons (c and d), respectively, that showed either very little or substantial Ih regardless of neurotransmitter phenotype. B, amplitude of time-dependent rectification was calculated for each neuron during voltage steps to −130 mV (b – a, as shown in Ad). C, time constants (tau) of tail currents from a single exponential fit of peak tail current during relaxations from −130 mV to −70 mV. C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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Some physiological properties distinguished GABAergic from non-GABAergic neurons quite accurately during whole-cell recordings in the present study but other previously suggested criteria did not. DAergic but not non-DAergic neurons in rat and guinea-pig VTA/substantia nigra have been reported to exhibit a slow depolarizing potential preceding action potential initiation during a step current injection (Grace & Bunney, 1984; Yung et al. 1991; Sarti et al. 2007). This slow depolarizing potential resembles the 4-aminopyridine-sensitive current in the ‘transient outward component’ in SNc reported by Silva et al. (1990). During whole cell recordings absence of a slow depolarizing potential was definitive for GABAergic neurons in the present study. All non-GFP neurons tested (n = 25) exhibited a slow depolarizing potential whilst none of the GAD-GFP neurons tested (n = 26) did. The absence or presence of a 4-aminopyridine-sensitive, slow depolarizing potential in the present study therefore provides a reliable criterion to distinguish DAergic from GABAergic neurons in VTA. Of course, this criterion can only be applied during whole cell recording when using intracellular solutions that do not disrupt K+ currents. Action potential duration was significantly briefer in GAD-GFP than non-GFP neurons, including those confirmed by post hoc immunohistochemistry to be TH positive. This is in agreement with intracellular micro-electrode recordings of presumed non-dopaminergic neurons in rats (Grace & Onn, 1989; Johnson & North, 1992a,b) and patch-clamp recordings in mice (Ford et al. 2006), but not by Margolis et al. (2006) in rats. However, there was some overlap of action potential durations between GAD-GFP and non-GFP cells, suggesting that whole-cell action potential duration alone is not a sufficient criterion to distinguish DAergic from GABAergic neurons in VTA. Similar differences were found for action potential rise time. Passive properties have also been reported to differ between TH positive and TH negative neurons in mouse (Ford et al. 2006) and rat (Margolis et al. 2006) VTA, but no difference in membrane conductance (from −60 mV to −80 mV) was found in the present study. Although the presence or absence of an I h current has been used widely to distinguish DAergic from non-DAergic neurons in midbrain SNc and VTA, (Lacey et al. 1989; Johnson & North, 1992b), the amplitude and kinetics of I h in TH positive neurons can vary greatly depending on projection targets (Ford et al. 2006), cellular chemistry (Neuhoff et al. 2002) and morphology (Sarti et al. 2007). Some TH negative VTA neurons have also been reported to exhibit large I h currents (Jones & Kauer, 1999; Margolis et al. 2006). In the present study, we confirmed a large range I h amplitudes and kinetics among VTA neurons. Here we show that the majority of GAD-GFP neurons exhibited only a very small I h amplitude and,

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when present, the decay kinetics tended to be rapid. Although many non-GFP neurons exhibited larger, slower I h currents, many did not, confirming that this criterion is not suitable to unequivocally distinguish DAergic from GABAergic VTA neurons. The large variable I h sizes in non-GFP VTA neurons in the present study may be due to neurons recorded from a wide range of anatomical coordinates, despite low number of neurons in each anatomical subfield. Anatomically distinct ‘groups’ of VTA DA neurons have different functional characteristics (e.g. Neuhoff et al. 2002; Ford et al. 2006; Sarti et al. 2007; Lammel et al. 2008; Margolis et al. 2008; Zhang et al. 2010) and have different efferent projections (e.g. Ford et al. 2006; Lammel et al. 2008; Margolis et al. 2008). In the present study, DA, but not DAMGO, inhibited all non-GFP neurons but some DA neurons have been reported to not express D2 autoreceptors (Lammel et al. 2008). The most likely explanation is that not many neurons sampled in the present study were medial to 3n/ml (see Fig. 4) where mesoprefrontal DAergic neurons that do not have postsynaptic D2 autoreceptors have been reported (Lammel et al. 2008). In conclusion, the present study has established that mouse GABAergic VTA neurons can be distinguished very reliably from DAergic neurons during electrophysiological recordings. During cell-attached recordings action potential durations of the two populations do not overlap. Distinct responses to μ-opioids and dopamine also distinguish the populations in the majority of neurons. During whole cell recordings, the presence or absence of a slow depolarizing potential is the most reliable criterion for distinguishing GABAergic and non-GABAergic neurons in VTA. Other properties such as action potential rise time and duration, and properties of I h currents can strengthen the classification but are not sufficient to reliably classify VTA neurotransmitter phenotype.

References Cameron DL, Wessendorf MW & Williams JT (1997). A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids. Neuroscience 77, 155–166. Ford CP, Mark GP & Williams JT (2006). Properties and opioid inhibition of mesolimbic dopamine neurons vary according to target location. J Neurosci 26, 2788–2797. Franklin KB & Paxinos G (1997). The Mouse Brain in Stereotaxic Coordinates. Academic Press. Garzon M & Pickel VM (2001). Plasmalemmal mu-opioid receptor distribution mainly in nondopaminergic neurons in the rat ventral tegmental area. Synapse 41, 311–328. Grace AA & Bunney BS (1984). The control of firing pattern in nigral dopamine neurons: single spike firing. J Neurosci 4, 2866–2876. C 2011 The Authors. Journal compilation C 2011 The Physiological Society

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Grace AA & Onn SP (1989). Morphology and electrophysiological properties of immunocytochemically identified rat dopamine neurons recorded in vitro. J Neurosci 9, 3463–3481. Johnson SW & North RA (1992a). Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12, 483–488. Johnson SW & North RA (1992b). Two types of neurone in the rat ventral tegmental area and their synaptic inputs. J Physiol 450, 455–468. Jones S & Kauer JA (1999). Amphetamine depresses excitatory synaptic transmission via serotonin receptors in the ventral tegmental area. J Neurosci 19, 9780–9787. Kawano M, Kawasaki A, Sakata-Haga H, Fukui Y, Kawano H, Nogami H & Hisano S (2006). Particular subpopulations of midbrain and hypothalamic dopamine neurons express vesicular glutamate transporter 2 in the rat brain. J Comp Neurol 498, 581–592. Korotkova TM, Ponomarenko AA, Brown RE & Haas HL (2004). Functional diversity of ventral midbrain dopamine and GABAergic neurons. Mol Neurobiol 29, 243–259. Lacey MG, Mercuri NB & North RA (1989). Two cell types in rat substantia nigra zona compacta distinguished by membrane properties and the actions of dopamine and opioids. J Neurosci 9, 1233–1241. Lammel S, Hetzel A, Hackel O, Jones I, Liss B & Roeper J (2008). Unique properties of mesoprefrontal neurons within a dual mesocorticolimbic dopamine system. Neuron 57, 760–773. Margolis EB, Lock H, Hjelmstad GO & Fields HL (2006). The ventral tegmental area revisited: is there an electrophysiological marker for dopaminergic neurons? J Physiol 577, 907–924. Margolis EB, Mitchell JM, Ishikawa J, Hjelmstad GO & Fields HL (2008). Midbrain dopamine neurons: projection target determines action potential duration and dopamine D2 receptor inhibition. J Neurosci 28, 8908–8913. Nair-Roberts RG, Chatelain-Badie SD, Benson E, White-Cooper H, Bolam JP & Ungless MA (2008). Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat. Neuroscience 152, 1024–1031. Neuhoff H, Neu A, Liss B & Roeper J (2002). Ih channels contribute to the different functional properties of identified dopaminergic subpopulations in the midbrain. J Neurosci 22, 1290–1302. Olson VG & Nestler EJ (2007). Topographical organization of GABAergic neurons within the ventral tegmental area of the rat. Synapse 61, 87–95. Prasad K & Richfield EK (2008). Sporadic midbrain dopamine neuron abnormalities in laboratory mice. Neurobiol Dis 32, 262–272.

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Sarti F, Borgland SL, Kharazia VN & Bonci A (2007). Acute cocaine exposure alters spine density and long-term potentiation in the ventral tegmental area. Eur J Neurosci 26, 749–756. Silva NL, Pechura CM & Barker JL (1990). Postnatal rat nigrostriatal dopaminergic neurons exhibit five types of potassium conductances. J Neurophysiol 64, 262–272. Steffensen SC, Svingos AL, Pickel VM & Henriksen SJ (1998). Electrophysiological characterization of GABAergic neurons in the ventral tegmental area. J Neurosci 18, 8003–8015. Tamamaki N, Yanagawa Y, Tomioka R, Miyazaki J, Obata K & Kaneko T (2003). Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J Comp Neurol 467, 60–79. Tecuapetla F, Patel JC, Xenias H, English D, Tadros I, Shah F, Berlin J, Deisseroth K, Rice ME, Tepper JM & Koos T (2010). Glutamatergic signaling by mesolimbic dopamine neurons in the nucleus accumbens. J Neurosci 30, 7105–7110. Ungless MA, Magill PJ & Bolam JP (2004). Uniform inhibition of dopamine neurons in the ventral tegmental area by aversive stimuli. Science 303, 2040–2042. Yamaguchi T, Sheen W & Morales M (2007). Glutamatergic neurons are present in the rat ventral tegmental area. Eur J Neurosci 25, 106–118. Yung WH, Hausser MA & Jack JJ (1991). Electrophysiology of dopaminergic and non-dopaminergic neurones of the guinea-pig substantia nigra pars compacta in vitro. J Physiol 436, 643–667. Zhang TA, Placzek AN & Dani JA (2010). In vitro identification and electrophysiological characterizaiton of dopamine neurons in the ventral tegmental area. Neurophamacology 59, 431–436.

Author contributions B.C. designed and performed the experiments, analysed and interpreted the data, drafted the manuscript and revised it critically for important intellectual content; Y.A. and S.M. performed and analysed immunohistochemical experiments; and M.C. contributed to conception, design and analysis of the experiments, drafting the article and revising it critically for important intellectual content.

Acknowledgements This study was supported by National Health and Medical Research Council of Australia (NH&MRC). M.J.C. is a NH&MRC Senior Principal Research Fellow (511914). Donation of GAD-GFP transgenic mice by Professor T. Kaneko is gratefully acknowledged.