Introduction

1. 0.81 ms 3.68 Hz B 2.17 ms On Off Current pulses Entrainment Mean = 1/ (mean firing rate) ISI Mean = 1 Var = 1.3 Counts 0 3 0 1 1 2 0 1 2 5 0 1 4 2 3 Spikes/mean ISI Neurophysiological characterization of the retrorubral field: focus on dopamine neurons of the A8 areaCA Mejias-Aponte1*and G Aston-Jones21Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia PA2Department of Neurosciences, Medical University of South Carolina, Charlestown, SC Introduction Results Firing regularity properties of RRF neurons 6 DA-like neurons are inhibited by cocaine The retrorubral field (RRF) contains 20% of the midbrain dopamine (DA) neurons (A8 nucleus). RRF efferents are intermingled among DA neurons of the ventral tegmental area (VTA) and substantia nigra (SN). They target limbic structures such as the amygdala and nucleus accumbens as well as the caudate. Cortical structures innervated by the RRF are mainly within the temporal lobe, including the piriform and entorhinal cortices, and the hippocampus. Behavioral studies in monkeys have shown that midbrain DA neurons, including the RRF, respond to reward predictors, primary rewards and reward value (Ljunberg et al., 1992; Schultz et al., 1993; Mirenowicz and Schultz, 1994; Tobler et al., 2005). The RRF DA neurons have historically been less studied than DA neurons of the VTA and SN. The present study aimed to phenotype DA neurons within the RRF using electrophysiological, histological and pharmacological tools. Distinguishing DA neurons from other RRF cells is the first step to understanding their physiology and drive further research on their function. 1 DA-like neurons can be distinguished based on spike width Wide-spike neurons are cells with action potential duration over 2 msec. Measuring their action potential from the start to the negative trough divided the population into 2 subgroups, UK-WS that peaks around 0.9 msec and DA-like that peaks at 1.2 msec. Short-spike neurons are cells with action potential duration less than 2 msec. These neurons cannot be differentiated by their spike width. Notice that wide-spike neurons did not differ in firing regularity. The UK-SS population had a greater percentage of irregular spiking neurons than any of the other groups (* p < 0.01, Fisher’s exact test). Similarly, UK-SS neurons showed a Fano factor above 1 indicating irregular discharge, a characteristic feature that was significantly greater than the other groups (**One-way ANOVA, F(3, 145) = 5.77 p < 0.001, Newman Keuls p < 0.01). The irregular discharge of UK-SS can be explained in part by their tendency for burst firing, which on average was higher than the other groups (*** % spikes in burst One-way ANOVA F(3,130) = 6.54 p < 0.001, Neuman-Keuls p < 0.01). DA-like neurons UK-WS neurons Methods 4 DA-like neurons were inhibited by footshock and was the group most often responsive to footshock Single-unit recordings were obtained from halothane anesthetized rats. Glass electrodes were lowered into the RRF using the following coordinates: 6.3-7.1 mm AP, 1.5-2.3 mm ML, 6.0-8.0 mm DV. Neurons were grouped according to firing rate and the width of the action potential (see diagram below). RRF neurons DA-like neuron UK-WS neuron < 2 ms whole spike width > 2 ms whole spike width 2 24% of RRF neurons are DA-like Both DA-like and UK-LS neurons responded to cocaine administration. The dotted line represents the time of cocaine injection (1 mg/kg, iv). DA-like neurons were inhibited by cocaine (≥ 50%, 4 of 5). UK-WS neurons were inhibited by cocaine in 5 of 7 cells, whereas the other 2 cells were excited. Short-spike neurons Wide-spike neurons DA-like neurons are represented by the red circles in the right lower quadrant. With the exception of black circle cells in the upper right quadrant that also have a spike width ≥ 1.1 msec, but with spontaneous firing rates over 10 Hz, RRF DA-like neurons fire under 10 Hz. The pie chart on the graph shows the proportion of each cell type relative to all neurons recorded. < 10 Hz > 10 Hz > 1.1 ms spike width up to the negative trough, > 10 Hz 7 UK-WS neurons do not express tyrosine hydroxylase, a marker for DA neurons < 1.1 ms spike width up to the negative trough Unknown short-spike neuron (UK-SS) GABA-like neuron A C DA-like neuron Unknown wide-spike neuron UK-WS Proportion of neurons that responded to footshock DA-like 2.17 ms In addition to firing rate, we analyzed firing regularity and bursting activity. The coefficient of variation (CV) was calculated by dividing the standard deviation by the mean interspike interval (ISI). The Fano factor was calculated by dividing the variance by the mean of the density discharge histogram (DDH). Regularity DDH was compared to a Poisson distribution with a mean equal to 1 using 2 goodness of fit. DDH different from Poisson Regular discharge variance < 1 Irregular discharge variance > 1 DDH not different from Poisson Random discharge Burst discrimination Threshold bin is the first DDH bin with at least 2 spikes/mean ISI where count is less than that of the following bin. If this criterion is not met, the threshold is the bin (≥ 2 spikes/mean ISI) having the smallest change in counts to the following bin. Threshold ISI is found by dividing mean ISI by the threshold bin number Percentage spikes in burst all spikes with an ISI ≤ to the threshold ISI divided by the total number of spikes X 100. Cellular properties of a UK-WS neurons juxtacellularly labeled with neurbiotinamide. A) Characteristics of the action potential. B) Entrainment of the cellular activity to juxtacellular current pulses. C) Absence of tyrosine hydroxylase staining (red) in the UK-WS neurons (green). None of six labeled neurons were positive for tyrosine hydroxylase. * The proportion of DA-like neurons that respond to footshock was significantly greater than the other groups, Fisher’s exact test, p < 0.05 Summary Firing characteristics of RRF neurons 3 DA-like neurons were inhibited by a D2 receptor agonist and recovered after a D2 receptor antagonist 5 • DA-like neurons • Spike width is a good criterion to distinguish DA-like neurons within RRF. • RRF is a heterogeneous region in which DA-like neurons comprise 24% of the spontaneously active neurons. • DA-like neurons were inhibited by footshock. The response can be evoked by stimulation of either the ipsilateral or contralateral paw. • DA-like neurons comprised the group most often responsive to footshock. • DA-like neurons were inhibited by the D2 DA receptor agonist quinpirole. Eticlopride, a D2 DA receptor antagonist, reversed the effect of quinpirole and produced excitation suggesting that DA-like neurons are under constant inhibition by DA. • DA-like neurons were inhibited by cocaine. The amount of inhibition is similar to what has been previously reported in VTA (Einhorn et al., 1988). • UK-WS neurons • A subgroup of UK-WS neurons can be distinguished from DA-like neurons by their excitatory response to footshock. • UK-WS neurons responded to a D2 agonist (quinpirole) and cocaine. Their responses can be either an inhibition or an excitation indicting that UK-WS neurons may comprise more that one type of neuron. Wide-spike neurons Discharge rates of DA-like neurons were uniform across a range of 0.2 to 6.5 Hz. The majority of UK-WS cells fire under 5 Hz with individual neurons discharging at rates as high as 38 Hz. Short-spike neurons Short-spike cells were segregated into two groups: < 10 Hz, and > 10 Hz (see division line). Previous studies had indicated that GABA neurons within midbrain DA areas discharge above 10 Hz (Steffensen et al., 1998). DA-like neurons UK-WS neurons The DDH was constructed by first subdividing the spike train into bin sizes equal to the mean ISI of the spike train. The number (i.e. density) of spikes that occurred during each bin was counted, and a frequency histogram was created based upon this discharge density. The DDH was also used to discriminate bursts (Kaneoke and Vitek, 1996). All cell groups were tested with a noxious stimulus (footshock, 10 mA, 0.5 Hz, 0.5 msec duration pulses). Wide-spike neurons were tested for the presence of the D2 DA receptor, and for responses to cocaine. A subset of wide-spike neurons were juxtacellularly labeled and examined for the presence of tyrosine hydroxylase, a marker for catecholamine neurons. References Schultz W, Apicella P, Ljungberg T (1993) J Neurosci. 13:900-13. Steffensen SC, Svingos AL, Pickel VM, Henriksen SJ. (1998) J Neurosci. 18:8003-15. Tobler PN, Fiorillo CD, Schultz W (2005) Science 307:1642-5. Conclusion Einhorn LC, Johansen PA, White FJ (1988) J Neurosci. 8:100-12. Kaneoke Y, Vitek JL (1996) J Neurosci Methods. 68:211-23. Ljungberg T, Apicella P, Schultz W (1992) J Neurophysiol. 67:145-63. Mirenowicz J, Schultz W. (1994) J Neurophysiol. 72:1024-7. The presence of the D2 autoreceptor was tested by systemic administration of the D2 agonist, quinpirole (quin, 100 µg/Kg), followed 2 minutes later by a D2 antagonist, eticlopride (100 µg/kg). The first dotted line indicates the time of quinpirole injection, and the second dotted line the time of eticlopride injection. DA-like neurons were inhibited > 50% of baseline discharge rate by quinpirole. Eticlopride reversed the effect of quinpirole and produced excitation in 4 of 5 cells suggesting that DA-like neurons are under constant inhibition by DA. UK-WS varied in their responses to quinpirole. Two neurons were inhibited, one was excited, and no effect was observed in one cell. Eticlorpide reversed the effect of quinpirole in only one cell. DA-like neurons can be distinguished from other neurons of the RRF. The best tools to identify DA-like neurons are the spike width and response to noxious stimulation. Future work needs to include histological verification of DA-like neurons juxtacellularly labeled to establish a definite phenotype. Acknowledgements Supported by PHS grants DA06214, DA017289 and DA016877