Review
Are you or aren’t you? Challenges associated with physiologically identifying dopamine neurons

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The dopamine system is involved in motivation, reward and learning, and dysfunction in this system has been implicated in several disorders, including Parkinson's disease (PD) and schizophrenia. Key progress in our understanding of its functions has come from extracellular in vivo electrophysiological recordings from midbrain dopamine neurons. Numerous studies have used a defined set of criteria to identify dopamine neurons electrophysiologically. However, a few recent studies have suggested that a minority population of non-dopamine neurons may not be readily distinguishable from dopamine neurons, raising questions as to the reliability of past findings. We provide an overview of the key findings related to this controversy and assess the criteria used for the electrophysiological identification of dopamine neurons in the substantia nigra pars compacta (SNC) and ventral tegmental area (VTA).

Introduction

Midbrain dopamine neurons located in the SNC and VTA (Figure 1a,b) play key roles in a broad range of behaviours, and are implicated in regulating goal-directed behaviour and response to reward. In addition, dopamine is believed to be central to several clinical disorders, including PD, schizophrenia, drug addiction and attention deficit hyperactivity disorder 1, 2, 3, 4, 5. Progress in our understanding of the role of the dopamine system in normal function and in disease states has been made on several fronts, including anatomical studies, neurochemical investigations and electrophysiological recordings. Indeed, crucial insights into their function have come from extracellular in vivo electrophysiological recordings. However, for such recordings to be applicable to the broad literature on the dopamine system, one must be confident that the neuron being recorded is indeed a dopamine-containing and -releasing neuron. Such an endeavour can be complicated when one must penetrate several millimetres into the brain with an electrode, and be able to segregate electrical signals coming from dopamine neurons from those arising from other cell types in the same brain regions (Figure 1c,d). For example, midbrain dopamine neurons are located within the mesencephalon in regions including the SNC and VTA, which also contain GABAergic neurons and glutamatergic neurons. Although dopamine neurons represent the majority [around 70%, although there is subregional variation; GABA around 30%; and glutamatergic 2–3% in VTA only (see below for more detailed discussion)] 6, 7, 8, 9, they are intermingled with neurons representing these other neurochemical types. Consequently, in order to draw valid conclusions relating to dopamine neuron activity, it is important to be confident that one is indeed recording from this specific neuron class.

To address this issue, a series of studies have established a method to identify putative dopamine neurons by their unique broad waveform characteristics, irregular or bursting firing pattern, and slow firing rate (Box 1). This approach has generated a substantial and influential body of work examining the rapid reward-coding properties of these neurons in primates and rodents (e.g. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). In addition, this has permitted the exploration of how dopamine neuron firing may be involved in disorders including PD [20], schizophrenia 21, 22 and addiction [23]. Although these criteria have proven sufficient to identify dopamine neurons in the SNC, in the VTA there have been reports of a minority of neurons (within the range 0–37%; average 11.6%) 24, 25, 26, 27, 28 that share some of these electrophysiological characteristics but could not be identified as dopaminergic using juxtacellular labelling and immunofluorescence for tyrosine hydroxylase (TH; the rate limiting enzyme in dopamine synthesis) 24, 25, 26, 28, 29. The suggestion that some recordings, once thought to have been made from dopamine neurons, may have mis-identified the neurons in question is an important issue because it raises doubt as to the comparability of these data with established neurochemical and behavioural investigations. This has implications for understanding, for example, how dopamine neurons respond to aversive events because some of these non-dopamine neurons are excited by such events (and may have been assumed to be dopaminergic in previous studies), which would not appear to be consistent with the suggestion that dopamine neurons are selectively activated by reward-related events (reviewed in [30]).

This issue has unfortunately generated confusion in the field about the reliability of criteria for the in vivo identification of dopamine neurons in both the VTA and SNC, and the relevance of previous literature that relied on such identification procedures. This also represents a significant challenge for those currently working on midbrain dopamine neurons. Moreover, a similar controversy has emerged concerning the electrophysiological criteria used for identifying dopamine neurons in brain slices 31, 32, 33. Here, we review the relevant literature and assess criteria used to identify dopamine neurons in the VTA and SNC. We suggest that, as initially defined, identification should be based on more than just action potential duration, and that relatively straightforward and reliable criteria for the identification of dopamine neurons in vivo in both SNC and VTA are available. We also discuss the identification of dopamine neurons during brain slice recordings, and some outstanding questions for future resolution. The issues raised here are illustrative of challenges faced when trying to elucidate in vivo neuronal function in the brain, where most regions contain intermingled neurochemically heterogeneous neurons.

Section snippets

In vivo identification of SNC dopamine neurons

Dopamine neurons were first identified in the SNC based on pharmacological criteria such as inhibition by dopamine receptor agonists and activation by dopamine receptor antagonists [11], loss of neurons with this electrophysiological phenotype after neurochemically specific lesions 11, 20, and antidromic activation from terminal regions [34]. Subsequent in vivo intracellular recording and labelling studies (see Glossary) in anaesthetised rats established that neurochemically-identified SNC

In vivo identification of VTA dopamine neurons

Similar to the SNC, extracellular recordings in the VTA report two types of electrophysiologically-distinct neuronal groups: broad-waveform slow-firing neurons (i.e. <10 Hz) and narrow-waveform fast-firing neurons (Figure 2d,e). This first type of neuron is typically assumed to be dopaminergic based on their similarity to SNC dopamine neurons, histochemical identification with intracellular staining [42], and recordings in in vitro brain slices, where neurochemical identity has been confirmed

Is the same identification issue present when recording in vitro?

Dopamine neurons can be recorded in vitro in midbrain slices (coronal, horizontal or sagittal) containing the VTA and SNC, and in this preparation also generally exhibit broad action potential waveforms and slow firing rates, similar to those seen in vivo (Figure 3). However, dopamine neurons are devoid of most of their inputs in brain slices owing to transaction of afferent fibres. As a result, dopamine neurons recorded in vitro do not exhibit burst firing and irregular firing observed in vivo

Identification of distinct subpopulations within the VTA and SNC

It is well established that there is diversity amongst SNC and VTA dopamine neurons with respect to their projection targets (e.g. striatal versus cortical 64, 65, 66), pharmacology (e.g. dopamine D2 receptor auto-inhibition 42, 54), molecular markers {e.g. calbindin-, G protein-gated inwardly rectifying K+ channel (Girk2)- and orthodenticle homeobox 2 (otx2) transcription factor-positive neurons [67]} and neuropeptide co-release (e.g. neurotensin [68]). Currently, there are no

Concluding remarks

The firing activity of midbrain dopamine neurons codes for reward- and punishment-related information, and changes in firing activity may contribute to the pathophysiology of several disorders, including schizophrenia and addiction. In most cases, studies have relied upon electrophysiological criteria for the identification of dopamine neurons in both the VTA and SNC. However, some confusion has arisen in the field concerning the reliability of electrophysiological criteria used for the

Acknowledgements

We thank Paul Bolam, Frederic Brischoux, Howard Fields and Elyssa Margolis for comments on earlier versions. Preparation of this article was supported by grant U120085816 from the UK Medical Research Council (MRC; to M.A.U), a University Research Fellowship from The Royal Society (to M.A.U.), and National Institutes of Health grants MH57440 and DA15408 (to A.A.G.).

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