Abstract
Brain regions typically contain intermixed subpopulations of neurons with different connectivity and neurotransmitters. This complicates identification of neuronal phenotypes in electrophysiological experiments without using direct detection of unique molecular markers. A prime example of this difficulty is the identification of dopamine (DA) neurons in the midbrain ventral tegmental area (VTA). Although immunocytochemistry (ICC) against tyrosine hydroxylase (TH) is widely used to identify DA neurons, a high false negative rate for TH ICC following ex vivo electrophysiology experiments was recently reported, calling into question the validity of comparing DA and non-DA VTA neurons based on post-hoc ICC. However, in whole cell recordings from randomly selected rat VTA neurons we have found that TH labeling is consistently detected in ∼55% of neurons even after long recording durations (range: 2.5–150 min). This is consistent with our prior anatomical finding that 55% of VTA neurons are TH(+). To directly estimate a false negative rate for our ICC method we recorded VTA neurons from mice in which EGFP production is driven by the TH promoter. All 12 EGFP(+) neurons recorded with a K-gluconate internal solution (as used in our rat recordings) were strongly labeled by TH ICC (recording duration 16.6±1.8 min). However, using recording electrodes with an internal solution with high Cl− concentration reduced the intensity of TH co-labeling, in some cases to background (recording duration 16.7±0.9 min; n = 10). Thus TH is a highly reliable molecular marker for DA neurons in VTA patch clamp recordings provided compatible microelectrode solutions are used.
Highlights
Powerful aspects of in vivo and ex vivo electrophysiological techniques include that they can reveal neural activity correlated with specific behaviors, sorting of neural properties by efferent and afferent connections, and sensitivity of particular circuit elements to endogenous and exogenous chemical stimulation
We first investigated whether recording in the same configuration that we previously used to compare the properties of DA and nonDA ventral tegmental area (VTA) neurons [1], but for a much shorter duration, would result in a higher detection rate of tyrosine hydroxylase (TH) in recorded neurons
When we recorded from EGFP(+) VTA neurons using the KCl-based internal solution described by Zhang et al [7], we found that the TH immunocytochemical signal was drastically degraded, and in some cases not discernable from background labeling (Figure 2D,E,F,G)
Summary
Powerful aspects of in vivo and ex vivo electrophysiological techniques include that they can reveal neural activity correlated with specific behaviors, sorting of neural properties by efferent and afferent connections, and sensitivity of particular circuit elements to endogenous and exogenous chemical stimulation. The strength of the interpretation of such studies depends on the extent and accuracy of the phenotypic information available for each recorded neuron. The more that is known about the circuit in which the recorded neuron participates and the neurotransmitters and neuromodulators it releases, the more informative electrophysiological observations become. Neurons in many brain regions, those in subcortical reticular structures, can be highly heterogeneous in neurotransmitter content and connectivity but similar in their electrophysiological properties. This makes it imperative to use molecular markers in conjunction with electrophysiology to fully understand how individual neurons function within defined circuits
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
