Brief Dopaminergic Stimulations Produce Transient Physiological Changes in Prefrontal Pyramidal Neurons

Abstract

In response to food reward and other pertinent events, midbrain dopaminergic neurons fire short bursts of action potentials causing a phasic release of dopamine in the prefrontal cortex (rapid and transient increases in cortical dopamine concentration). Here we apply short (2 sec) iontophoretic pulses of glutamate, GABA, dopamine and dopaminergic agonists locally, onto layer 5 pyramidal neurons in brain slices of the rat medial prefrontal cortex (PFC). Unlike glutamate and GABA, brief dopaminergic pulses had negligible effects on the resting membrane potential. However, dopamine altered action potential firing in an extremely rapid (<1s) and transient (<5min) manner, as every neuron returned to baseline in less than 5-min post-application. The physiological responses to dopamine differed markedly among individual neurons. Pyramidal neurons with a preponderance of D1-like receptor signaling respond to dopamine with a severe depression in action potential firing rate, while pyramidal neurons dominated by the D2 signaling pathway respond to dopamine with an instantaneous increase in spike production. Increasing levels of dopamine concentrations around the cell body resulted in a dose dependent response, which resembles an “inverted U curve” (Vijayraghavan et al., 2007), but this effect can easily be caused by an iontophoresis current artifact. Our present data imply that one population of PFC pyramidal neurons receiving direct synaptic contacts from midbrain dopaminergic neurons would stall during the 0.5 sec of the phasic dopamine burst. The spillover dopamine, on the other hand, would act as a positive stimulator of cortical excitability (30% increase) to all D2-receptor carrying pyramidal cells, for the next 40 seconds.

1. Introduction

Transient release of dopamine (DA) in the brain is thought to occur upon expected food reward following positive reinforcers such as juice, or any cue that predicts the availability of the reward (Schultz, 2002). As a result of the behaviorally relevant stimuli DA neurons exhibit a burst of activity, which constitutes a phasic component of DA release in the brain (Ljungberg et al., 1992). This “phasic mode” of DA release is thought to be important for proper functions of the prefrontal cortex, PFC (Williams and Goldman-Rakic, 1995; Schultz, 1998; Heien and Wightman, 2006). Since it is technically impossible to precisely control dopamine release during elaborate experimental paradigms by simply shocking the midbrain (Lewis and O'Donnell, 2000), researchers have mimicked transient DA release by iontophoretic application of dopamine via glass pipettes inserted into the cortex of awake monkeys (Sesack and Bunney, 1989; Matsumura et al., 1990; Williams and Goldman-Rakic, 1995; Sawaguchi, 2001; Vijayraghavan et al., 2007).

Although these experiments highlight the importance of DA in PFC, the cellular mechanisms by which DA modulates PFC function, specifically in terms of its influence on single cell activity, remain largely unknown. Single unit recordings in awake, behaving animals are not ideally suited to analyze the cellular mechanisms of dopaminergic effects. It is still not clear if the observed DA-ergic modulations of neuronal firing rate were mediated through modulation of synaptic inputs (synaptic excitability), modulation of neuronal membrane excitability (intrinsic excitability), or both. Additionally, continuous applications of exogenous dopaminergic drugs (≥ 60 sec) (Vijayraghavan et al., 2007) cannot mimic the phasic dopamine activity that lasts shorter than 1 sec (Ljungberg et al., 1992).

Here, we performed whole-cell measurements in rat PFC brain slices to determine the basic physiological effects of brief (phasic) DA release on layer 5 pyramidal neurons. The synaptic influences on neuronal membrane potential were silenced pharmacologically, allowing us to focus solely on intrinsic membrane properties. Dopaminergic drugs were delivered locally on the cell body for 2 sec. By using a local drug release, we avoided the confounding influence of bath application of dopaminergic drugs reflected in the sustained, indiscriminant and simultaneous activation of all dopamine-responsive elements in the brain tissue. Miniature changes in membrane potential that arose from transient DA iontophoresis were evaluated, and compared with glutamate and GABA ejections, using identical experimental settings (i.e. stimulus current intensity, duration, shape of the drug pipette and its position and distance in respect to the cell body). Our data show the quality, quantity, and precise temporal dynamics of dopamine-induced changes on action potential output of a major projection neuron of the mammalian PFC, a layer 5 pyramidal cell. The observed transient changes in the neuronal action potential output may be occurring in vivo during behavioral paradigms that are characterized by phasic dopaminergic signaling, and also during in vivo experiments with microiontophoresis of dopamine (Vijayraghavan et al., 2007). A preliminary report has been presented in abstract form (Moore et al., 2009).

2. Results

Neuron subtype

Within the cortical layer 5, individual neurons were identified by their large pyramidal shaped cell body (∼15 μm in diameter) and by the presence of long apical dendrites extending towards the pial surface (Milojkovic et al., 2005). In response to suprathreshold current steps these cells exhibited modest spike frequency accommodation, often after an initial spike doublet. This firing pattern corresponds to the “intrinsic bursting” cells described by Yang et al., 1996.

Glutamate and GABA

Local iontophoretic application of excitatory neurotransmitter glutamate (current intensity range: 20 – 100 nA) on layer 5 pyramidal neurons of the rat prefrontal cortex issued graded depolarizations of the membrane potential (Vr), as previously reported (Langomoen and Hablitz, 1981; Milojkovic et al., 2005). In each neuron tested with glutamate (n=7), the depolarization response was strong enough to elicit action potential firing followed by a depolarization block (Fig. 1B, glutamate). On average the peak (maximal) depolarization elicited by glutamate iontophoresis at 100 nA intensity of iontophoretic current was 29.4 ± 5.5 mV (n=7), Fig. 1C, diamonds). Iontophoretic application of the inhibitory neurotransmitter, GABA resulted in a small depolarization of the neuronal membrane (5 – 10 mV, (n=7), Fig. 1 B, C), similar in amplitude to those previously reported in the rat neocortex (8.2 ± 0.9 mV)(Gulledge and Stuart, 2003; Szabadics et al., 2006). The depolarizing action of GABA surprised many researchers because cortical inhibitory interneurons use GABA as a primary neurotransmitter (Krnjevic and Schwartz, 1967). Gulledge and Stuart (2003) have provided an elegant explanation for this paradox. At resting membrane potential a significant fraction of the ion flux through GABA_A receptor channels is comprised by bicarbonate ions (equilibrium potential = -12 mV), leading to depolarizing GABAergic responses, irrespective of the site of GABA receptor activation (soma or dendrite). These GABA-mediate depolarizing potentials should not be mistaken with excitation. GABA is still an inhibitory transmitter because the equilibrium potential of GABA_A receptors (combined chloride and bicarbonate equilibrium) is more negative than the action potential voltage threshold; hence the ensuing “shunting” inhibition effectively prevents AP generation in pyramidal neurons (Staley and Mody, 1992). Note that in our experiments local stimulations with GABA generated robust depolarizations (up to 10 mV) but no AP firing (Figure 1B, GABA).

Figure 1. Local neurotransmitter application.

A) Schematic of the experimental paradigm in this and all the following figures. B) Change in membrane potential (Vm) following glutamate, GABA, or dopamine iontophoretic application on layer 5 pyramidal neurons in the rat medial prefrontal cortex. In contrast to glutamate and GABA, dopamine has a negligible effect on resting voltage. Bottom trace marks the timing and duration (2 s) of the iontophoretic current pulse. C) Average voltage response to glutamate (diamonds), GABA (squares), and dopamine (triangles) at different intensities of iontophoretic current, from 20 to 100 nA. “n” – indicates number of neurons per experimental group.

Dopamine

We iontophoretically applied DA onto the pyramidal cell with the drug pipette at the same location as for glutamate and GABA experiments (Fig. 1A), and we used the same intensities of iontophoretic current (in the range 20 – 100 nA, fixed duration = 2 sec). Unlike glutamate or GABA, dopamine had very little effect on the membrane potential, Fig. 1B, Dopamine). Even with maximal current pulses of 100 nA DA only elicited 0.5 ± 0.05 mV change in the membrane potential (n=33 Fig. 1C, triangles). Once again, the size and position of the drug-filled pipettes, as well as the intensity of the iontophoretic currents were identical in the case of glutamate, GABA and dopamine, which allowed us to produce a fair, side-by-side comparison (Fig. 1B, C). These data show that dopamine action on the resting membrane potential of layer 5 pyramidal neurons in the medial prefrontal cortex is negligible.

Artifacts induced by the stimulus current

Iontophoretic current pulses delivered in close vicinity of the cell (Fig. 2A, schematic drawing) are apt to introduce experimental artifacts. We analyzed membrane potential fluctuations at two time points (Fig. 2D). One time point (“1.8 s”) was chosen to cover physiological response during the actual iontophoretic pulse (Fig. 2D, 1.8 s). Note that in classic papers the neuronal firing rate is assessed while the iontophoretic current pulse is continuously on (Sesack and Bunney, 1989; Matsumura et al., 1990; Williams and Goldman-Rakic, 1995; Sawaguchi, 2001; Vijayraghavan et al., 2007). The second time point (“2.2 s”) was chosen to assess the membrane response immediately after the cessation of iontophoretic pulse, when electric field is no longer imposed onto the neuronal membrane (Fig. 2D, 2.2 s). In all experiments except the one shown in Fig. 2A, dopamine was loaded into the drug application pipette (In Pipette). Brain slices were either perfused with regular ACSF (Fig. 2A, B) or with dopamine antagonists dissolved in ACSF (Fig. 2C, D, E; In Bath). Each experimental condition is thus characterized by dopamine or no dopamine (ACSF only) inside the application pipette (In pipette) and one or two or no drugs in the bath (In Bath), which is schematically displayed in the top inset of each figure panel (Fig. 2).

Figure 2. Miniature membrane potential changes upon brief dopaminergic stimulus.

A) Local application of ACSF alone (no drugs included inside the application pipette) reveals an experimental artifact at current intensities of 200 and 600 nA. B) Same as in A except dopamine is included in the application pipette. C) Dopamine-induced changes in resting membrane potential in the presence of D1 dopamine receptor antagonist, SCH23390 (SCH) in the bath. Arrow indicates hyperpolarization after the termination of iontophoretic current pulse. (D) DA inside the application pipette and D2 DAr antagonist sulpiride (SP) in the bath. Two time points (1.8 and 2.2 sec from the onset of iontophoretic current) were used in the analysis. E) The perfusion of SCH23390 and sulpiride together also reduces the current artifact. F) Change in membrane potential (Vm) at 80 nA (TOP) and 600 nA (BOTTOM) measured at 2.2 sec time point. Measurements at 1.8 s time point are not shown. Horizontal lines represent the group average and s.e.m. Circles indicate a single response from each cell in the group. Group sizes: ACSF/ACSF (n=14 cells); DA/ACSF (n=33 cells); DA/SCH (n=9 cells): DA/SP (n=9 cells); DA/SCH+SP (n=6 cells). Asterisks marks significant difference from DA/ACSF group, p < 0.05.

To characterize the stimulation artifact being generated in our experimental paradigm we omitted dopamine from the glass pipette (ACSF only). At current amplitudes smaller than 100 nA (20 – 80 nA) the microiontophoresis of ACSF alone (no drug) had no effect on the neuronal membrane potential (Fig. 2A, 40 nA and 80 nA). However, an obvious stimulation artifact was introduced into the system starting at 200 nA, and its amplitude at 600 nA reached on average 2.61 ± 0.26 mV (mean ± s.e.m., n = 14) when measured at time point 1.8 sec from the onset of iontophoresis (Fig. 2A).

In the next set of measurements we iontophoretically applied DA onto the pyramidal cells at the same distance, same current intensities (from 20 to 600 nA) and same pulse duration (Fig. 2B, Iont. Pulse) as in the previous measurement (Fig. 2A). Iontophoretic DA application up to 100 nA (duration 2 sec) changed the somatic membrane potential by < 0.5 mV, similar to ACSF (Fig. 2, compare A and B, 40 – 80 nA). At higher current pulses (600 nA) the average response to dopamine near the end of the iontophoretic pulse (time point “1.8 sec”) was 1.56 ±0.47 mV (n=33 cells). Changes in resting membrane potentials obtained with DA were on average less positive than those obtained when DA was omitted from the ACSF. However, a Student's t-test failed to detect significant difference between two groups (p=0.062). Bath applications of selective D1 and D2 receptor antagonists, SCH23390 and sulpiride, either alone (Fig. 2C, D) or together (Fig. 2E), failed to alter DA effect on resting membrane potential during the iontophoretic pulse (time point 1.8 s, 20 – 600 nA, p>0.05).

In some experiments we noticed changes in resting membrane potential that occurred after the cessation of iontophoretic current pulse (Fig. 2C, arrow). We quantified these data by measuring the change in membrane potential at a second time point (“2.2 s”), which occurs when electric field is no longer imposed onto the neuronal membrane (Fig. 2D). These measurements are plotted in Fig. 2F. The depolarizing effect of dopamine (Fig. 2F, DA/ACSF) was significantly attenuated only when both SCH and SP were included in perfusion (Fig. 2F, 80 nA, asterisk, p=0.014). For both stimulation intensities (80 nA and 600 nA) the raster plots (Fig. 2F) show that in the DA group (DA/ACSF) data points (grey circles) are scattered in a wide range. Some pyramidal neurons respond to DA iontophoresis with depolarization, while others respond by hyperpolarization of the somatic membrane. Due to a great variability within “DA/ACSF” group the Student's t-test failed to detect any significant difference between this group and bath applications of DAr antagonists either alone (DA/SCH, DA/SP) or together (DA/SCH+SP; p>0.05). These results indicate that at higher stimulation intensities (>200 nA) a considerable experimental artifact is being generated with microiontophoresis apparatus, which is very difficult to discern from the pharmacological effects of exogenous dopamine.

Synaptic factor

Previous studies have highlighted both the direct effect of dopamine on layer 5 pyramidal neurons and the indirect effect of dopamine through activation of GABAergic interneurons (Ferron et al., 1984; Godbout et al., 1991; Pirot et al., 1992; Gulledge and Jaffe, 2001; Gorelova et al., 2002). Next, we tested the direct effect of dopamine on the membrane potential in the presence of synaptic blockers, NMDA, AMPA, and GABA receptor antagonists (APV, DNQX, and Picrotoxin, respectively). Acute slices were bathed in 5 μM APV, 10 μM DNQX, and 20 μM Picrotoxin for at least 20 minutes prior to local DA application. Application of DA elicited identical responses on the resting membrane potential in the presence (n = 10) or absence of synaptic blockers (n=33, Fig. 3A). We also measured changes to the membrane potential caused by microiontophoresis of selective D1 receptor agonist (SKF38393) and D2 dopamine receptor agonist (quinpirole) in the presence of synaptic blockers, and compared these changes to those obtained in regular ACSF (Control, Fig. 3B, C). In all three experimental groups (dopamine, SKF and quinpirole), and at all stimulation intensities (20 – 600 nA), we failed to detect any significant differences between measurements performed with synaptic blockers (grey) or without (black) synaptic blockers (Fig. 3; p>0.05). These data strongly indicate that the observed changes in the neuronal resting membrane potential were not mediated via synaptic circuits.

Figure 3. DA-induced change in resting membrane potential (Vr) is independent of synaptic inputs.

(A) Effects on Vr during a DA pulse were measured in both normal ACSF solution (control, black) and ACSF solution containing synaptic blockers (Syn. Block, grey). Synaptic block cocktail includes: DNQX [10 μM], APV [5 μM], and Picrotoxin [20 μM]. Introduction of synaptic blockers does not alter neuronal responses to dopamine (A), or SKF38393 (B), or quinpirole (C).

DA effect on membrane excitability

To determine if brief dopaminergic stimulation alters the intrinsic membrane excitability we locally applied dopamine via an iontophoretic current pulse (Fig. 4A), while the cell was current-clamped to fire a train of action potentials (Fig. 4B). In the beginning of each experiment the amplitude of the depolarizing current pulse was adjusted to elicit a firing frequency of 8 – 10 Hz (duration 10 s). After the baseline firing frequency was established with two control recordings (only one shown in Fig. 4C), DA was iontophoretically applied to the cell soma from a drug pipette positioned approximately 25 μm away (Fig. 4A). The dopamine iontophoresis pulse duration was set at 2 seconds, and it occurred amidst the 10 second depolarizing current step (Fig. 4B, iontophoretic pulse). Eight gradually increasing intensities of DA iontophoretic current (20 nA – 600 nA) were explored in 8 consecutive sweeps (30 sec between sweeps).

Figure 4. Brief dopamine pulse alters intrinsic membrane excitability.

A) DIC image of a layer 5 pyramidal cell and two glass micropipettes: “Patch” for whole-cell recordings and “DA” for dopamine iontophoresis. Scale, 20 μm. In this and all remaining figures the synaptic block cocktail includes: DNQX [10 μM], APV [5 μM], and Picrotoxin [20 μM]. B) Top trace shows a train of action potentials induced by depolarizing current. Bottom trace shows the timing and duration (2 s) of the dopamine iontophoretic pulse. All measurements of instantaneous frequency (Inst. Freq.) were taken during drug application (grey box, During). C) Each experiment began by establishing the baseline firing frequency. Iontophoretic application of ACSF alone does not affect action potential firing frequency at lower stimulation intensities 20 – 100 nA. Dopamine application, on the other hand, first increases (20 – 100 nA) then decreases action potential firing (200 - 600 nA). D) Instantaneous frequency during stimulus (During) was normalized against baseline frequency (thick horizontal line in C) and averaged across all cells in the data set for each stimulation intensity. In one data set application pipettes were filled with ACSF (grey), while in the other DA was included (black). E) ACSF-subtracted DA response reveals a 10% increase in instantaneous frequency peaking at 80 nA. F) Sequential application of ACSF and DA in the same cell reveals clear differences between neuronal responses to ACSF and DA. Asterisks, p ≤ 0.05.

Because of the observed current artifact on the resting membrane potential during ACSF application (Figs 2 and 3) we first sought to determine if the iontophoretic current pulse alone was able to alter the action potential output of the cell. The iontophoretic application of ACSF alone (no drugs) failed to alter AP firing at the lower current range (20 – 200 nA, Fig. 4B, ACSF). At higher intensities (400 and 600 nA) the ACSF-filled electrode strongly reduced AP firing during the iontophoretic application (Fig. 4C grey; n=15). We averaged data from 45 dopamine-treated cells and plotted them in Fig. 4D, black line. An average of 15 ACSF-treated cells (grey) is superimposed for direct comparison. T-test analysis of the two groups failed to detect any statistically significant differences at any given stimulation intensity (20 – 600 nA) between DA and ACSF groups. To eliminate current stimulation artifacts we subtracted the ACSF response from the DA response (Fig. 4E). The artifact-corrected data suggest that on average dopamine initially increases the firing frequency during DA release of up to 100 nA. At stimulation intensities higher than 100 nA, DA has no net effect on cortical AP firing, based on the average obtained from many neurons (Fig. 4C, E), but see Fig. 5.

Figure 5. Variable response to DA puff among layer 5 pyramidal neurons.

A) During local iontophoretic application of DA (grey box), cells displayed either no response to dopamine (No Change, top), an increase (Increase, middle), or a decrease in action potential firing (Decrease, bottom). B) Individual responses from all cells in data set (n=45 cells) are plotted to emphasize a great degree of variability. Based on a greater than 20% change in firing frequency at 40 and 60 nA, these 45 cells were separated into 3 categories: 1) No Change (medium grey), 2) Increase (black), and 3) Decrease (light grey). C) The average responses from three categories are plotted and labeled accordingly. ACSF is treatment with ACSF only (no drug inside application pipette). “n” indicates the group size. Asterisks, p ≤ 0.05 compared to ACSF group.

In a subset of cells (n=12) we applied first ACSF and later DA to the same cell. Direct comparison of recordings obtained with ACSF and DA application on the same pyramidal cell (paired T-test) revealed the dopamine-induced increase in action potential firing which was statistically significant for stimulation intensities within the range from 20 to 100 nA (Fig. 4F, asterisks). These results suggest that during DA application, while the neuron is depolarized (generating repetitive action potential firing) the dopaminergic stimulation initially increases intrinsic excitability. Once the concentration of DA reaches a certain value it no longer increases excitability but rather depresses the intrinsic firing of the pyramidal neurons in prefrontal cortex. This trend, characterized by a gradual increase in AP firing rate at low intensities of stimulation followed by a decrease in AP firing at higher intensities of stimulation is reminiscent of the inverted-U dose-response (Vijayraghavan et al., 2007). It must be noted here that stimulus artifact (microiontophoresis current) has a strong potential to suppress firing of cortical pyramidal neurons at higher stimulation intensities (Fig. 4D, ACSF), therefore every experiment involving DA iontophoresis across a wide range of iontophoretic intensities, would eventually generate an inverted-U dose-response curve.

Variability among layer 5 pyramidal neurons (Fig. 5B)

During DA application approximately half (49%) of the cells examined (22 of the 45 cells) did not respond to the drug; the response was less than a 20% change from baseline firing rate established prior to dopamine application (Fig. 5C, No Change). Another population of layer 5 pyramidal cells, comprising 35.5% of the complete data set (16 out of 45) showed a strong increase in AP firing frequency, which peaked around 100 nA (Fig. 5C, increase) resembling the ACSF-subtracted DA response (Fig. 4E). The smallest representation (15.5 %) of cells strongly decreased firing frequency in response to dopamine. In this group, at higher stimulation intensities, we often observed a complete block of AP firing (200 to 600 nA, Fig. 5C, Decrease). ANOVA analysis performed on 3 subsets of pyramidal neurons (No Change, Increase and Decrease) failed to detect any statistically significant differences in respect to (i) membrane potential; (ii) membrane input resistance; or (iii) number of APs evoked by standard current pulse (p>0.05). These data show that PFC layer 5 pyramidal neurons constitute a homogeneous group in respect to basic physiological properties, yet three distinct groups emerge in respect to dopaminergic modulation of membrane excitability (Fig. 5). In the next series of experiments we test the hypothesis that three kinds of responses to dopaminergic stimulation are product of differential expression of dopaminergic receptor subfamilies among individual pyramidal cells.

Receptors

Dopamine receptors are divided into two subfamilies, D1-like and D2-like (Civelli et al., 1993). D1-like DA receptors (D1 and D5 subtypes) are positively coupled to the intracellular production of cyclic-AMP. D2-like DA receptors, including D2, D3, and D4 receptor subtypes, on the other hand, cause a decrease in cAMP level. Dopamine receptor expression among layer 5 pyramidal neurons in the prefrontal cortex is highly heterogeneous. Cortical pyramidal cells express either D1-like DA receptors, D2-like receptors, or both on the same cell (Vincent et al., 1993; Bergson et al., 1995; Gaspar et al., 1995; Vincent et al., 1995; Santana et al., 2008). Two dopamine receptor families (D1-like and D2-like) have different affinities for DA, with D2 receptors having a higher affinity causing it to be stimulated at lower concentrations (lower stimulation intensities 20 nA – 100 nA, Figs 4 and 5). Likewise, D1 receptors have lower affinity for dopamine, and therefore are more likely to dominate cellular responses at higher DA concentrations (higher stimulation intensities). In the next series of experiments the setup was identical to that shown in Fig. 4A, B, except glass pipettes were filled with selective agonists instead of dopamine.

Selective D1 DAr agonist SKF38393

Brief application of selective D1 dopamine receptor agonist SKF38393 at lower iontophoretic intensities (20 – 40 nA) caused a slight increase in action potential firing rate (n=15, Fig. 6A, SKF, 40 nA). At higher stimulation intensities SKF38393 depressed firing and in some cells (n=10/15) produced a complete block in AP firing (Fig. 6A, SKF, 600 nA). An averaged response across all neurons in this data series (n=15) revealed an overall suppressive action of D1 receptor stimulation on neuronal excitability throughout a wide range of stimulation intensities (Fig. 6B, top). Interestingly the average suppression in AP firing rate caused by the D1 agonist (Fig. 6B, top) was similar in amplitude and dose-dependence to that observed for dopamine (Fig. 5C, Decrease). The major difference between the two drugs, dopamine and SKF38393, was in the duration of the effect. DA-induced suppressions in AP firing occurred only during the course of iontophoretic application (Fig. 4C, Dopamine, 600 nA), while SKF38393 effect regularly persisted several seconds after the ejection (Fig. 6A, SKF 600 nA). This difference may be due to a faster clearance of DA from the brain tissue by the means of faster diffusion, degradation and uptake of the natural compound (dopamine) compared to synthetic compound (SKF). Apart from the differences in the duration of the effect, the peak amplitudes of DA and SKF effect were quite comparable (compare Fig. 5C, Decrease and Fig. 6B). Saturation of the curves (Fig. 5C and Fig. 6B) suggests that within the range of stimulation intensities used (20 – 600 nA) we have achieved the maximal effect (maximal suppression of excitability) physiologically possible for both drugs dopamine and SKF38393. The amplitude of the effect varies from neuron to neuron but on average and across the entire population of PFC layer 5 pyramidal neurons a 60% reduction in the AP firing rate was measured. If we disregard individual differences among neurons and monitor a circuit or ensemble of PFC pyramidal neurons as a whole, for example, the strongest D1 dopaminergic stimulus would be expected to cut the population activity down to 40%. The dopaminergic D1 modulation is very rapid as it takes less than a second to develop and manifest in the AP firing rate (Fig. 6A). Given the local application of drugs via glass micropipette positioned at the cell body these data also suggest that suppression in neuronal excitability is the result of D1-like receptor stimulation located on and near the cell body of layer 5 pyramidal cells (Fig. 4A).

Figure 6. Effects of dopamine receptor specific agonists on intrinsic membrane excitability.

A) Changes in instantaneous frequency were recorded during application of the D1 receptor agonist SKF38393 (left) or the D2 receptor agonist quinpirole (right). B) Changes in instantaneous firing frequency (Inst. Freq.) were normalized against Control values and averaged across all cells in the data series application of SKF38393 (top, n=15) or quinpirole (bottom, n=21). Release of SKF38393 onto the cell soma decreases action potential firing, while release of quinpirole increases the frequency of action potential firing.

Selective D2 DAr agonist quinpirole

Brief application of the D2 receptor agonist, quinpirole, locally on the somata of PFC layer 5 pyramidal neurons (n=21 neurons) caused a rapid increase in AP firing rate (Fig. 6A, quinpirole). The average amplitude of the effect grew across the stimulation range 20 – 80 nA, reaching its maximum at 100 nA. At stimulation intensities greater than 100 nA (200 – 600 nA) the D2-induced increase in firing rate remained at maximum (plateau effect), suggesting that the cells' response has been exhausted. In other words, the experiments covered a physiologically plausible range for D2 dopaminergic stimulation.

The plot shown in Fig. 6B, Quipirole, includes the entire set of quipirole treated cells (n=21). Next we selected a group of quipirole-treated neurons with an increase greater than 20%, as previously performed on the DA group (Fig. 5C, Increase). The average amplitude of increase for a subset of quinpirole-teated neurons with a strong effect (n = 9) was 35 ± 0.07 % which was very similar to the DA-treated subset with increasing firing rates (39 ± 0.1 %, n=16, Fig. 4). These data suggest that DA-induced increase in firing rate (Fig. 5C) was most likely mediated through D2-like dopamine receptors located on, or near the cell body of PFC layer 5 pyramidal neurons. The neuronal response to D2 receptor stimulation was very fast, as changes in instantaneous AP firing frequency were regularly observed in less than one second from the onset of drug stimulus (Fig. 6A, Quipirole, 40 nA).

Duration of the dopamine-induced change in intrinsic excitability

In the previous experiments we have seen that dopamine-receptor stimulation produced rapid modulations of AP firing; within one second from the onset of the drug stimulus (Figs 4C, 5A, and 6A). Next we asked how long the DA-stimulated cell would remain in an altered state. The design of the experiment was identical to that shown in Fig. 4A. The intensity of stimulation was set to 60 nA because at this intensity the majority of pyramidal neurons respond with an increase in AP firing rate. Following a brief and local application of dopamine onto the cell (Fig. 7A1, 60 nA), the source of the iontophoretic current was turned off. In subsequent recording trials a standard depolarizing step has been repeated every 30 seconds (Fig. 7A1, Wash). On average, cells recovered (returned to baseline firing) within the first 270 seconds of recording (Fig. 7A2). Recovery from either SKF38393-induced depression (Fig. 7B) or quinpirole-induced excitation (Fig. 7C) followed a nearly identical time course to that observed for dopamine (Fig. 7A). These data show that dopaminergic modulation of membrane excitability is a transient event. Following a brief dopaminergic stimulus at the cell body the production of AP in a target pyramidal is altered for a short period of time; less than 270 seconds.

Figure 7. Rapid recovery of dopamine-mediated effect on membrane excitability.

A₁) In the beginning of each experiment a baseline firing rate is established (Control). Upon local exposure to dopamine (DA, 60 nA), pyramidal neurons increase the production of action potentials. Subsequent recordings (taken every 30 seconds) are used to monitor the washout of the DA-induced effect. A₂) A plot of normalized AP firing frequency versus time reveals that firing rates return to their baseline (Control) level in ∼270 seconds, on average (n=32 cells). B_1,2) Same experiments as in the previous panel except instead of DA, a selective D1 receptor agonist SKF38393 was applied on the cell body (n=8). C_1,2) Same as above except D2 receptor agonist quinpirole was used in the application pipette (n=15).

Neuronal excitability right after the phasic DA signal

In the present study so far, all quantifications of DA-induced changes in neuronal excitability were made during the actual application of dopamine, at time window dubbed “During” (Fig. 4B). Unlike glutamate or GABA that work directly through membrane channels (ionotropic mechanism), dopamine works through second messenger systems (metabotropic mechanism) that may have a longer lag time before a full onset, and longer duration after the onset. To investigate the delayed action of dopamine receptor stimulation we quantified neuronal firing rates in a time window that takes place 2.5 sec after the cessation of the DA iontophoretic pulse (Fig. 8B, grey window “Delay-1”). The positive effect of DA on neuronal firing rate was considerably stronger several seconds after DA application (Fig. 8B, Delay-1), compared to DA action during the drug application (Fig. 8A, During). For example, at 200 nA stimulus intensity the average neuronal firing rates are depressed by ∼20 % during DA application (Fig. 8A, During), and enhanced by ∼30%, only a couple of seconds later (Fig. 8B, Delay-1). Stimulus current artifacts (Fig. 4C, ACSF) do not affect Delay-1 period, and this could explain a large difference between the During and Delay-1 time windows.

Figure 8. Delayed response to a brief DA pulse.

A) Top-Schematic: Changes in instantaneous frequency were quantified during application of DA in the time window marked by grey rectangle. Bottom-Graph: DA and ACSF plots are same data as in Fig. 4D. In this and the following panels “Control” marks a group of cells (n=12) in identical experimental paradigm (10 sweeps), except the microiontophoresis apparatus was turned OFF throughout the experiment. B) Top-Schematic: Grey window marks the time window chosen for quantification of neuronal firing rates. This time window occurs 2.5 s after the cessation of DA pulse. Bottom-Graph: Delayed changes in instantaneous firing frequency (Delay-1) show an increase in firing rate through the entire range of stimulation intensities. C) Top-Schematic: The time window chosen for quantification of neuronal firing rates is 40 sec after the cessation of DA pulse (Delay-2). Note that Delay-2 includes a period of 30 sec without injection of depolarizing current. Bottom-Graph: Delayed-2 response is similar to Delay-1 in previous panel. Data was normalized to the same designated time window (grey window: During, Delay-1, or Delay-2) in two control sweeps that precede each paradigm.

While waiting for DA effect to envelop in this experimental paradigm (Fig. 8B) the neurons were kept in depolarized state by a 10 s-long direct current injection. Next we ask what would happen if neurons were allowed to return to resting potential while waiting for DA-mediated pathways to envelop. Time interval between 2 successive experimental trials (sweeps) was set to 30 sec (Fig. 8C, 30 s). We quantified neuronal firing in the time period dubbed “Delay-2”, which occurs approximately 40 seconds after the cessation of the dopamine iontophoretic pulse. Group data (Fig. 8C, Graph) revealed that neuronal firing rate remain elevated for at least 40 seconds, even if 30 out 40 seconds were spent in rest. The amplitude of elevation [∼30 %] was identical at 2.5 and 40 seconds; compare Delay-1 and Delay-2 (Fig. 8, compare B and C). These data are in good agreement with the previous set of experiments shown in Fig. 7A, which indicates that 30 - 60 seconds post DA stimulus the neuronal firing rate is elevated by ∼30%, on average. Note that entirely different neurons comprise data groups featured in Figures 7A and 8C. In summary, immediately following a phasic DA-ergic stimulus the PFC layer 5 pyramidal neurons experience an overall (∼30 %) increase in the intrinsic membrane excitability, which lasts at least 40 seconds post-stimulus (Fig. 8), and then returns to baseline in the next 200 seconds (Fig. 7A).

3. Discussion

In this study we attempted to mimic the phasic dopaminergic signaling in prefrontal cortex (Schultz, 2002) in an in vitro setting. We eliminated synaptic communication and focused on dopamine-induced changes on intrinsic membrane excitability alone. We eliminated the stimulation of remote dendritic regions by applying dopamine precisely at the cell body of positively identified layer 5 pyramidal neurons. In this way we restricted our analysis to activation of dopamine receptors at, and near the cell body, a principle neuronal compartment for generation of action potentials (Kole et al., 2008). Unlike glutamate and GABA neurotransmitter release, which can strongly depolarize the somatic membrane, DA has a minimal effect on the resting membrane potential (Figs 1, 2 and 3). Despite its inability to change the resting potential, DA can rapidly alter membrane excitability while the neuron is in the depolarized state, firing trains of action potentials (Figs 4, 5 and 6). The DA-induced change in membrane excitability is dependent on: (1) the intensity of the dopaminergic stimulus; and (2) the type of dopamine receptor activated (D1-like or D2-like; Fig. 6). By applying dopaminergic stimuli of different intensities to each pyramidal neuron we aimed to characterize the spectrum of physiological responses in PFC that might occur in behavioral scenarios characterized by different rates of dopamine neuron discharge (Shultz 2000). Our data show that dopaminergic modulation of neocortical membrane excitability is a transient event, with fast onset (<1 s) and short duration (∼4 minutes, Fig. 7).

Methodological Considerations

Dopaminergic neurons project their axons into the PFC from the ventral tegmental area (VTA) (Van Eden et al., 1987). A short (sub-second) burst of action potentials, like the one occurring upon food or liquid reward (Ljungberg et al., 1992), is likely to cause a transient dopamine release from axon terminals invading the PFC (Goldman-Rakic et al., 1989; Verney et al., 1990; Benes et al., 1993; Sesack et al., 1998b; Sesack et al., 1998a). In support of this notion rapid changes in cortical dopamine concentrations, over the course of several seconds, have been directly detected by electrochemical methods (Rebec et al., 1997; Richardson and Gratton, 1998). But exactly how changes in cortical dopamine tone affect cortical neuronal circuits and cortical signal processing remains a topic of active debate (Gorelova and Yang, 2000; Henze et al., 2000; Gulledge and Jaffe, 2001; Maurice et al., 2001; Seamans et al., 2001; Gonzalez-Burgos et al., 2002; Gao et al., 2003; Tseng and O'Donnell, 2005).

Stimulation of dopaminergic receptors in the prefrontal cortex in vivo, either by dopamine released by VTA stimulation or exogenously applied via pipette, yielded important information on the effect of rapid dopamine signaling on action potential firing rates (Bernardi et al., 1982; Thierry et al., 1986; Godbout et al., 1991; Pirot et al., 1992; Sesack et al., 1995b; Lewis and O'Donnell, 2000; Floresco and Grace, 2003). However, in vivo studies of dopaminergic modulation of neuronal excitability are burdened with several technical and conceptual problems. First, it is difficult to control the ambient natural fluctuations of dopamine tone occurring during the course of the experiment. Dopamine levels rapidly fluctuate in the cerebral cortex at any given period of time, and are influenced by a number of intrinsic and extrinsic factors (Schultz, 2002). Hence, in vivo studies are by default being performed on neurons that are exposed to a mixture of both natural dopamine and artificially applied dopaminergic agents, in unknown proportions and unknown temporal order. For example, accidental phasic midbrain activity may either precede or follow the artificial dopamine pulse. Second, in vivo stimulation of dopamine receptors either by local iontophoresis or electrical shock to the VTA has poor spatial precision, resulting in activation of an unknown set of dopamine receptors distributed somewhere along the complex dendritic tree of layer 5 pyramidal neurons. Furthermore, VTA stimulation indiscriminately engulfs massive numbers of pyramidal and non-pyramidal neurons residing in the immediate vicinity, and far away from the neuron under study. Hence, the VTA stimulation paradigm is not ideally suited to distinguish between “network” and “single-cell” effects of the dopaminergic signal. Third, electrical shocks to the VTA cause the release of neurotransmitters other than dopamine in the PFC (Carr and Sesack, 2000), which makes genuine dopamine effects difficult to isolate (but see (Tecuapetla et al., 2010)).

In vitro brain slice preparation, on the other hand, allows better control of neurotransmitter release, and a greater variety of experimental tools for studying the effect of dopaminergic stimulation on neuronal excitability (Hoffman and Johnston, 1999; Gao et al., 2003; Gulledge and Stuart, 2003; Rosenkranz and Johnston, 2006). The majority of in vitro studies relied on bath application of dopaminergic drugs (Law-Tho et al., 1994; Geijo-Barrientos and Pastore, 1995; Gulledge and Jaffe, 1998; Zhou and Hablitz, 1999). Introduction of drugs into the perfusion line results in a very slow, ramp-like, gradual increase in drug concentrations which takes minutes to develop. A more troubling deficiency of the bath application approach is the very long duration of the dopaminergic stimulus, also on the order of minutes. Yet, the most troubling aspect is massive and indiscriminate stimulation of absolutely all dopamine-receptive elements in the brain slice, including all types of pyramidal neurons, inhibitory interneurons, glia, and blood vessels. Such a slow-ramping, massive, indiscriminate and prolonged stimulation more closely resembles tonic DA signaling and is unlikely to mimic the delicate anatomical and physiological aspects of the organization of the dopaminergic system in the PFC in response to behavior-related phasic stimulation (Goldman-Rakic et al., 1992; Lewis et al., 1998; Seamans and Yang, 2004; Goto and Grace, 2007). Because VTA-to-PFC communication is in large part composed of discrete DA synaptic contacts onto dendrites and somata of specific pyramidal neurons (Goldman-Rakic et al., 1989) and distribution of these contacts is highly non-uniform across cortical lamina I-VI (Descarries et al., 1987; Gaspar et al., 1995), spatially-restricted (local) dopaminergic stimulation of individual cells (Fig. 4A) is a more direct experimental approach to examine the basic physiological consequences of phasic dopaminergic signaling in a well defined cellular compartment (e.g. cell body, Fig. 4A).

Here we used microiontophoresis and whole-cell patch clamp recordings in brain slices to monitor changes in physiological properties of positively identified layer 5 pyramidal neurons. This experimental approach brought 5 benefits: (1) Fast application and visual monitoring of the success of drug ejection; (2) Precise targeting of the cellular compartment of interest (cell body); (3) Elimination of synaptic influences; (4) Precise detection of stimulation artifacts; and (5) Monitoring of the fast onset and offset of drug-induced changes in neuronal excitability by recording membrane potential transients rather than relying on spike count alone (Sesack and Bunney, 1989; Matsumura et al., 1990; Williams and Goldman-Rakic, 1995; Sawaguchi, 2001; Vijayraghavan et al., 2007).

(1) Fast Application

In order to better mimic phasic dopaminergic activity two important parameters of DA-ergic stimulus, duration and timing were precisely controlled by a computer driven electrical pulse. This is in stark contrast to the experimental design used by Vijayraghavan et al. (2007), where iontophoretic application of dopaminergic drugs was continuous during the entire behavioral test (minutes). Under infrared DIC video-microscopy we carefully observed the tips of our drug-application pipettes (Fig. 4A). The iontophoretic pulse ejects a jet of drug-rich solution, which causes a ripple through the brain tissue. When the drug application pipette becomes clogged the ripple is absent. Continuous visual monitoring of the glass micropipettes allowed us to have superb control over the status of drug delivery. Clogged pipettes were replaced with fresh ones. The data obtained with clogged pipettes were discarded from analysis.

(2) Precise Targeting of cellular compartment

Electron microscopy studies reveal that dopaminergic axons make direct synaptic contacts on cell bodies of cortical pyramidal neurons (Goldman-Rakic et al., 1989; Smiley and Goldman-Rakic, 1993; Sesack et al., 1995a). Furthermore, immunostainings have shown that both families of dopamine receptors (D1- and D2-like) are expressed on the somatic plasma membrane (Vincent et al., 1993; Bergson et al., 1995). The axon-soma junction is the main integration point of neocortical pyramidal neurons where APs are initiated (Kole et al., 2008), therefore dopaminergic modulation of this compartment is likely to have a strong impact on the neuronal AP firing rate. Using infrared video microscopy (Fig. 4A) and focal application via a glass micropipette we released the dopaminergic drugs directly onto the soma, while avoiding the confounding effects, which may arise from DA-ergic stimulation of the remote neuronal compartments, or remote cells. Furthermore, we can continuously monitor the DA pipette tip and neuronal soma during the actual experimental measurement (Fig. 4A), thus we could assure consistent stimulus location and amplitude.

(3) Absence of network driven activity

Cells in our data set were not driven by network activity, but rather by the strictly regulated depolarizing current injected into the cell under current clamp. In our system the synaptic influences were blocked by glutamate and GABA antagonists (APV, DNQX, and picrotoxin). The quieted network activity allowed us to focus solely on the role of dopamine receptor activation on action potential output in PFC layer 5 pyramidal neurons.

(4) Determine and Eliminate Experimental Artifacts

One of the caveats of iontophoretic application is a current artifact, caused by the direct effect of iontophoretic current on the nearby neuronal membrane. We spent considerable experimental resources to evaluate the contamination of our data by experimental artifacts (Figs 2, 3, and 4). By measuring changes to the membrane potential (Fig. 2) and intrinsic excitability (Fig. 3) while delivering ACSF alone (no drug), we were able to quantify the introduced artifact at each applied current intensity, and eliminate it in the off-line analysis (subtraction) to reveal the pure drug response (Fig. 4E).

(5) Fast temporal dynamics of the dopaminergic modulation

In previous studies on dopamine-induced changes in AP firing rate, the precise onset of the effect was not systematically analyzed or, the means of dopamine application were too slow to properly address this question. It was recently reported that inhibitory effects of DA on AP firing rate and neuronal input resistance (R_in) were evident within 3 minutes of application (Gulledge and Jaffe, 2001). In our present study, we regularly observed a significant change in the action potential firing rate within the first second of dopamine release (Figs 4-6). These sub-second responses of PFC pyramidal neurons to dopaminergic stimulus match the dynamics of sub-second outburst in firing of dopaminergic neurons in vivo (Ljungberg et al., 1992). Our current experimental design also revealed that this change in neuronal excitability is transient and upon ceasing dopaminergic stimulus the neuronal production of AP readily returns to baseline values (within 270 seconds on average). These data suggest that phasic dopamine activity is able to instantaneously alter firing of individual pyramidal neurons during the behavioral or computational task at hand (Schultz, 2002). Following the outburst of phasic dopamine activity the PFC pyramidal neurons quickly return to their baseline excitability (Fig. 7).

Functional impact

Dopaminergic signaling in the PFC assumes two operational regiments: a constant but low level (low concentration) DA signal, often dubbed “ambient DA”, is occasionally interrupted by fast and strong (high concentration) DA transient, which is known as “phasic DA signal” (Schultz, 2002). Based on present in vitro data we think that phasic DA signal is poised to perturb action potential generation in the major projection neurons of the prefrontal cortex, layer 5 pyramidal cells, by direct action on the soma-axon compartment. Phasic DA-ergic signals do not perturb resting membrane potential (Figs 2 and 3) and their effect can only be revealed if the recipient pyramidal cells are actively firing action potentials (Figs 4-6). It must be emphasized here that we often encountered PFC Layer 5 pyramidal neurons that did not respond to dopamine (Fig. 5), so that the present conclusions (below) apply only to a subset of DA-sensitive pyramidal neurons.

Based on single unit recordings from midbrain DA-ergic neurons (Ljungberg et al., 1992) the duration of cortical DA release should be in the order of 1 sec. In general, a short duration of a biological signal often implies the rapid onset of its effect. In the present study we specifically examined the physiological changes that occur in pyramidal neurons during a brief (2 second) DA pulse (Figs 1 – 7), and found that such brief stimulus can either increase or decrease firing rates, depending on the balance between D1-like and D-2 like pathways. Pyramidal neurons with a preponderance of D1-like receptor signaling respond to phasic DA stimulus with a severe depression in action potential firing rate, while pyramidal neurons dominated by the D2-signaling pathway respond to dopamine with an instantaneous increase in spike production. D2 receptors have a greater affinity for DA than their D1 counterparts, and for this reason, the D2-like family of DA receptors is more likely to get activated by low-amplitude DA signals. This may explain the overall increases in neuronal firing rates at lower intensities of DA stimulus (20 nA – 100 nA, Fig. 4). At higher intensities of DA-ergic stimulation (200 nA – 600 nA) the D1-like receptors, which are more numerous in neocortex than D2 receptors (Lidow et al., 1991; Gaspar et al., 1995), take over, and such D1-dominated event produces a complete block in AP firing (Fig. 5A, Bottom trace; Fig. 6A, Bottom trace). From the aspect of cortical information processing one scenario begins to emerge. A subset of Layer 5 pyramidal neurons with a dominant D2 pathway would be selectively activated during ambient and/or low-amplitude phasic DA signals. Pyramidal neurons with a dominant D1 pathway would decrease firing during the same low-amplitude phasic DA signal. High-amplitude phasic DA signals, on the other hand, have a potential to stall the entire PFC circuit for the short duration of the phasic DA-neuron burst. Pyramidal neurons that exit from DA-induced depression tend to be more excitable (Fig. 8, see also (Gulledge and Jaffe, 1998)). Our measurements indicate that several seconds after cessation of a phasic DA signal the overall excitability of PFC is enhanced by approximately 30% above baseline, for the next 40 seconds (Fig. 8C). In fairness, the complete decay of DA-induced effect takes ∼270 seconds (Fig. 7A2), but this could be an exaggeration due to in vitro experimental conditions. In physiological settings, in vivo, with intact cerebral circulation and active uptake of DA, this effect is likely to be shorter (Chergui et al., 1994). In both cases, 40 s or 270 s, the transient nature of physiological changes that take place in PFC upon a phasic DA signal fits the idea that one phasic DA signal is devoted to one cognitive task at hand (Ljungberg et al., 1992; Kosobud et al., 1994; Gurden et al., 1999; Schultz, 2002; Lisman and Grace, 2005).

Emerging scenario

Some, but not all, pyramidal cells receive direct synaptic contacts on their cell bodies (Goldman-Rakic et al., 1989). The actual concentration of DA in the synaptic cleft has never been measured, however it can be assumed that it extends into a millimolar range (Grace, 1991). Our experiments have established that strong DA-ergic input to the soma-axon compartment produces a severe block of action potential firing (Figs 4 to 6). These data implies that population of PFC pyramidal neurons receiving direct synaptic contacts from midbrain DA-ergic neurons would stall during the 0.5 sec phasic DA burst (Ljungberg et al., 1992). The spillover DA, on the other hand, would act as a positive stimulator of membrane excitability to all D2-receptor carrying pyramidal cells in the neighborhood, for the next 40 seconds.

4. Methods and Materials

Brain slice and electrophysiology

Sprague Dawley rats (P19 – 32) were anesthetized with isoflurane, decapitated, and the brains removed with the head immersed in ice-cold, artificial cerebrospinal fluid (ACSF), according to the animal protocol approved by the Center for Laboratory Animal Care, University of Connecticut Health Center. Artificial cerebrospinal fluid (ACSF) contained (in mM) 125 NaCl, 26 NaHCO₃, 10 glucose, 2.3 KCl, 1.26 KH₂PO₄, 2 CaCl₂ and 2 MgSO₄, pH 7.4; oxygenated continuously with a mixture of 95% O₂ and 5% CO₂. For blocking glutamatergic and GABAergic inputs 10 μM DNQX, 5 μM APV and 20 μM Picrotoxin were added to the ACSF solution. Coronal slices (300 μm) were taken from the frontal lobes anterior to the genu of corpus callosum. Slices were incubated for 1 hour at 36°C and then stored at room temperature prior to recordings. Acute brain slices were transferred to an Olympus upright microscope and gravity perfused with aerated ACSF at 34°C. Whole-cell recordings were made from visually identified layer 5 pyramidal neurons, on the medial part of the slice, as depicted in (Gulledge and Jaffe, 1998). Patch pipettes (5 – 7 MΩ) were filled with intracellular solution containing (in mM) 135 K-gluconate, 2 MgCl₂, 3 Na₂-ATP, 10 Na₂-phosphocreatine, 0.3 Na₂-GTP and 10 HEPES (pH 7.3, adjusted with KOH). Current clamp recordings were made using Multiclamp 700A (digitized using Digidata Series 1322A at 10 kHz) and Clampfit 9.2 software (Molecular Devices). Only cells with a membrane potential more hyperpolarized than -50 mV, and action potential amplitudes exceeding 80 mV (measured from the baseline) were included in this study.

Experimental paradigm

All measurements were performed in current-clamp mode. A series of command steps (-200 to +700 pA) in 100 pA intervals (duration 200 ms) was applied to verify the electrophysiological phenotype of the selected pyramidal neurons. All neurons in the present data set belong to an “intrinsic bursting” cell type (Yang et al., 1996). Following the physiological identification of the cell, the drug of choice (glutamate, GABA, dopamine, SKF38393, or quinpirole) was loaded into a 4 MΩ glass pipette and a continual “retention” current pulse of ±5 nA was used to prevent leakage from the pipette until drug release. Intrapipette concentrations of all drugs (glutamate, GABA, dopamine, SKF38393, or quinpirole) were 10 mM. For Control experiments ACSF alone was loaded into the glass pipette. Dopaminergic drugs were dissolved on the day of the experiment, to a final concentration of 10 mM with a pH of 4 – 4.5. A motorized micromanipulator (M-285, Sutter) was used to position the tip of the drug-application pipette 20 – 25 μm from the soma. Drugs were released by an iontophoretic current pulse (Microiontophoresis Dual Current generator 260, World Precision Instruments). We regularly used infrared video microscopy to monitor the success of the iontophoretic pulse ejection.

Trains of action potentials were evoked by a single depolarizing current step (duration 10 sec) repeated every 30 sec for a total of 10 trials. Current intensities were adjusted separately for each cell, so that they evoked a baseline firing frequency of 8 – 10 Hz (100 – 300 pA). After 2 baseline measurements of action potential firing frequency, the drug of choice was locally applied by iontophoresis onto the cell soma for 2 sec, at varying amplitudes of current in the following sequence: 20, 40, 60, 80, 100, 200, 400, and 600 nA. Selective D1 and D2 receptor antagonists were bath applied at following concentrations: SCH23390 (20 μM) or sulpiride (40 μM) for 20 minutes prior to DA application.

Data analysis

Whole-cell measurements were analyzed offline using Clampfit 9.2 (Axon Instruments). Maximal change in resting membrane potential was measured in the interval 1.8 – 2.5 sec after the onset of the drug pulse. The baseline resting membrane potential was measured 2 sec prior to drug application. The effect of dopamine on intrinsic membrane excitability was determined by the instantaneous AP firing frequency. Instantaneous frequency was calculated by measuring the inter-spike interval using a built in function of Clampfit. Numerical analysis and graph plotting was done in Excel. The measurements of AP firing rate are presented in relative terms; as percent change relative to control (baseline) measurement obtained before drug application. All error bars in figures and text were reported as the mean ± s.e.m. A statistical significance (p≤0.05) was determined using ANOVA analysis in SigmaStat 3.2.

Definition of “Excitability”

The term “excitability” is often used in physiological studies without proper explanation. In the present study the membrane excitability of a neuron is defined as the number of action potentials evoked by a standard current pulse delivered directly into the cell body. For example, an increase in the number of evoked action potentials is interpreted as “increase in intrinsic membrane excitability”.

Abbreviations

PFC

Prefrontal cortex

DA

Dopamine

DAr

Dopamine receptors

AP

Action potential

ACSF

Artificial cerebrospinal fluid

GABA

Gamma aminobutyric acid