Abstract

Whereas the entorhinal cortex (EC) receives profuse dopaminergic innervations from the midbrain, the effects of dopamine (DA) on γ-Aminobutyric acid (GABA)ergic interneurons in this brain region have not been determined. We probed the actions of DA on GABAA receptor-mediated synaptic transmission in the EC. Application of DA increased the frequency, not the amplitude, of spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) recorded from entorhinal principal neurons, but slightly reduced the amplitude of the evoked IPSCs. The effects of DA were unexpectedly found to be mediated by α1 adrenoreceptors, but not by DA receptors. DA endogenously released by the application of amphetamine also increased the frequency of sIPSCs. Ca2+ influx via T-type Ca2+ channels was required for DA-induced facilitation of sIPSCs and mIPSCs. DA depolarized and enhanced the firing frequency of action potentials of interneurons. DA-induced depolarization was independent of extracellular Na+ and Ca2+ and did not require the functions of hyperpolarization-activated (Ih) channels and T-type Ca2+ channels. DA-generated currents showed a reversal potential close to the K+ reversal potential and inward rectification, suggesting that DA inhibits the inward rectifier K+ channels (Kirs). Our results demonstrate that DA facilitates GABA release by activating α1 adrenoreceptors to inhibit Kirs, which further depolarize interneurons resulting in secondary Ca2+ influx via T-type Ca+ channels.

Introduction

The entorhinal cortex (EC) is considered as the gateway mediating the majority of connections between the hippocampus and other cortical areas. Sensory inputs from olfactory structures, parasubiculum, perirhinal cortex, claustrum, and amygdala converge onto the superficial layers (layers II/III) of the EC, which sends dense projections onto the hippocampus; the axons of the stellate neurons in layer II of the EC form the perforant path that innervates the dentate gyrus and Cornu Ammonis (CA)3, whereas those of the pyramidal neurons in layer III form the temporoammonic pathway that synapses onto the distal dendrites of pyramidal neurons in CA1 and the subiculum (Steward and Scoville 1976; Witter, Naber, et al. 2000; Witter, Wouterlood, et al. 2000). Furthermore, neurons in the deep layers of the EC (layers V/VI) relay a large portion of hippocampal output projections back to the superficial layers of the EC and to other cortical areas (Witter et al. 1989). The EC is part of a network that participates in the consolidation and recall of memories (Haist et al. 2001; Squire et al. 2004; Dolcos et al. 2005; Steffenach et al. 2005). Neuronal pathology and atrophy of the EC are potential contributors to Alzheimer's disease (Hyman et al. 1984; Kotzbauer et al. 2001) and schizophrenia (Falkai et al. 1988; Arnold et al. 1991; Joyal et al. 2002; Prasad et al. 2004). Moreover, the EC is closely involved in the induction and maintenance of temporal lobe epilepsy (Spencer and Spencer 1994; Avoli et al. 2002).

Catecholamines including dopamine (DA) and norepinephrine are neurotransmitters or neuromodulators involved in the modulation of a variety of physiological functions such as working memory (Phillips et al. 2008; Sara 2009) and neurological and psychiatric disorders, including Parkinson's disease, addiction, schizophrenia, bipolar disorder, Huntington's disease, attention deficit hyperactivity disorder, and Tourette's syndrome (Beaulieu and Gainetdinov 2011; Kurian et al. 2011). DA activates 5 types of G protein-coupled receptors that can be classified as D1- (D1 and D5) and D2-like (D2, D3, and D4) receptors (Beaulieu and Gainetdinov 2011), whereas norepinephrine interacts with α1, α2, β1, β2, and β3 adrenergic receptors. However, evidence suggests that there are promiscuous interactions among dopaminergic and adrenergic receptors. For example, DA has been shown to activate α1 (Leedham and Pennefather 1986; Rey et al. 2001; Cornil et al. 2002; Zhang et al. 2004; Lazou et al. 2006; Lin et al. 2008), α2 (Leedham and Pennefather 1986; Cornil et al. 2002), and β (Rajfer et al. 1988; Anfossi et al. 1993; Lee et al. 1998; Ouedraogo et al. 1998) adrenergic receptors, whereas norepinephrine activates D2 dopaminergic receptors (Robbins et al. 1988). At least 4 major dopaminergic pathways have been identified in the mammalian brain; the nigrostriatal, mesolimbic, mesocortical, and tuberoinfundibular tracts that originate from the dopaminergic neurons in the substantia nigra, ventral tegmental areas, arcuate nucleus, and periventricular area of the hypothalamus, respectively. Like other cortical regions, the EC receives profuse dopaminergic innervation mainly from the ventral tegmental areas in the midbrain (Akil and Lewis 1993). Similarly, the EC also receives prominent noradrenergic projections from the locus coeruleus (Fallon et al. 1978; Palkovits et al. 1979; Wilcox and Unnerstall 1990). Consistent with the anatomical dopaminergic and noradrenergic innervations of the EC, the EC also expresses dopaminergic receptors such as D1- (Savasta et al. 1986; Huang et al. 1992; Tarazi et al. 1999) and D2-like (Richfield et al. 1989; Weiner et al. 1991; Hemby et al. 2003; Rivera et al. 2008) receptors and adrenergic receptors including α1 (Wilcox and Unnerstall 1990), α2 (Unnerstall et al. 1984, 1985; Boyajian et al. 1987), and β (Booze et al. 1993) receptors. Functionally, DA increases Na+ channel currents (Rosenkranz and Johnston 2007), inhibits the excitability of pyramidal neurons (Rosenkranz and Johnston 2006; Mayne et al. 2013), and modulates excitatory synaptic transmission (Pralong and Jones 1993; Stenkamp et al. 1998; Behr et al. 2000; Caruana et al. 2006; Caruana and Chapman 2008) and plasticity (Caruana et al. 2007; Hamilton et al. 2010) in the EC. Application of norepinephrine in the EC inhibits glutamatergic transmission (Pralong and Magistretti 1994, 1995) and neuronal excitability (Xiao, Deng, Rojanathammanee, et al. 2009) via activation of α2 receptors and facilitates GABAergic transmission via the activation of α1 receptors (Lei et al. 2007). However, the effects of DA on inhibitory synaptic transmission and GABAergic interneurons are elusive, although DA has been shown to slightly depress evoked IPSPs in the EC (Pralong and Jones 1993). In this study, we thoroughly examined the effects and the underlying mechanisms of DA in GABAergic transmission in the EC. Our results showed that DA increased the frequencies of spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs), but slightly depressed the amplitude of evoked IPSCs (eIPSCs). Further investigation revealed that DA augmented the frequencies of sIPSCs and mIPSCs not by DA receptors, but by the activation of α1 adrenergic receptors. Determination of the underlying ionic and signaling mechanisms indicated that functions of the inward rectifier K+ channels (Kirs) and the T-type Ca2+ channels were required for DA-mediated facilitation of GABAergic transmission.

Materials and Methods

Slice Preparation

Horizontal brain slices (400 µm) were cut using a vibrating blade microtome (VT1000S, Leica, Wetzlar, Germany) from 14- to 21-day-old Sprague-Dawley rats as described previously (Deng and Lei 2006, 2007; Xiao, Deng, Rojanathammanee, et al. 2009; Deng, Xiao, Jha, et al. 2010; Ramanathan et al. 2012).

Recordings of GABAA Receptor-Mediated sIPSCs, mIPSCs, and eIPSCs

Whole-cell patch-clamp recordings in voltage- or current-clamp mode were made from stellate neurons in layer II or interneurons in layer III of the EC as described previously (Deng et al. 2006; Wang et al. 2011, 2012) unless stated otherwise. The recording electrodes were filled with (mM) 100 cesium gluconate, 0.6 ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 5 MgCl2, 8 NaCl, 2 ATPNa2, 0.3 GTPNa, 40 4-(2-Hydroxyethyl piperazine-1-ethanesulfonic acid (HEPES), and 1 QX-314 (pH 7.3). The extracellular solution comprised (mM) 130 NaCl, 24 NaHCO3, 3.5 KCl, 1.25 NaH2PO4, 1.5 MgCl2, 2.5 CaCl2, and 10 glucose, saturated with 95% O2 and 5% CO2 (pH 7.4). To record GABAA receptor-mediated sIPSCs, the external solution was supplemented with dl-2-Amino-5-phosphonopentanoic acid (dl-APV) (50 µM) and 6,7-Dinitroquinoxaline-2,3-dione (DNQX) (10 µM). Synaptic currents were recorded at a holding potential of +30 mV. mIPSCs were recorded by including tetrodotoxin (TTX) (0.5 µM) in the above external solution. eIPSCs were recorded from stellate neurons using the same internal and external solution by placing a stimulation electrode (a patch-clamp recording pipette filled with oxygenated extracellular solution) locally (∼200 μm from the recorded neuron). Data were filtered at 2 kHz, digitized at 10 kHz, and acquired on-line using the pCLAMP 9 (Clampex) software (Axon Instruments). The recorded sIPSCs and mIPSCs were subsequently analyzed by Mini Analysis 6.0.1 (Synaptosoft, Inc., Decatur, GA, USA). Each detected event was inspected visually to exclude obvious artifacts before analysis. The threshold for detection was set to 3 times the standard deviation of the noise as recorded in an event-free stretch of data. Mean amplitude, frequency, cumulative amplitude, and frequency histograms were calculated by this program. Because the basal frequency of the events varied considerably for individual cells, we normalized the average of the frequency of the events recorded for 5 min before the application of DA for better comparison. The recorded eIPSCs were analyzed by pClamp 9 (Clampfit).

Recordings of Resting Membrane Potentials, Action Potentials, and Holding Currents From Interneurons in Layer III

Resting membrane potentials (RMPs), action potentials (APs), and holding currents (HCs) were recorded from interneurons in layer III of the EC with the intracellular solution containing (in mM) 100 potassium gluconate, 0.6 EGTA, 5 MgCl2, 8 NaCl, 2 ATPNa2, 0.3 GTPNa, phosphocreatine 7, and 33 HEPES (pH 7.3 adjusted with KOH). As described previously (Lei et al. 2007; Deng and Lei 2008; Xiao, Deng, Yang, et al. 2009; Deng, Xiao, Lei, et al. 2010), interneurons were initially identified morphologically. The sizes of interneurons were generally smaller compared with the principal neurons in layer III. The shapes of interneurons could be bipolar, spindle, or ovoid. Their orientation was typically perpendicular to the axis of the surrounding principal cells in layer III and parallel to the pial axis of the slice. The properties of interneurons were further confirmed electrophysiologically because they showed fast spikes, whereas the principal neurons in layer III demonstrated slow spikes. We waited for 10–15 min after the establishment of whole-cell configuration to record stable responses. For the experiment involving N-methyl-d-glucamine (NMDG), the extracellular NaCl concentration was replaced by the same concentration of NMDG and HCl was used to adjust pH to 7.4. Current–voltage curves were constructed from the interneurons in layer III. K+-gluconate internal solution was used and the extracellular solution was supplemented with (µM) 0.5 TTX, 100 CdCl2, 200 NiCl2, 10 DNQX, 50 dl-APV, and 10 bicuculline. Current–voltage relationship was obtained by using a ramp protocol from −110 to −50 mV. Because the maximal effect of DA usually occurred at 5–10 min, we compared the current–voltage curves recorded before and when the maximal effect of DA was observed. Stock DA solution at 100 mM was initially prepared, aliquoted, and frozen till use. For each cell, 15 µL of the frozen DA stock solution was freshly dissolved in 15 mL of the extracellular solution and then applied to the cells in about 8 min to prevent oxidation of DA. We never observed noticeable change of the color of the solution, suggesting that there was no apparent oxidation of DA by this method.

Data Analysis

Data are presented as the means ± SEM. The concentration–response curve of DA was fit by Hill equation: I = Imax × {1/[1 + (EC50/[ligand])n]}, where Imax is the maximum response, EC50, the concentration of ligand producing a half-maximal response, and n, the Hill coefficient. Student's paired or unpaired t-test or analysis of variance was used for statistical analysis as appropriate. Statistical analysis was performed using Origin 7 and GraphPad Prism 4. P-values are reported throughout the text and significance was set as P < 0.05. For sIPSC cumulative probability plots, events recorded for 2 min before DA application and 2 min of the maximal effect of DA were selected. Same bin sizes (25 ms for frequency and 2 pA for amplitude) were used in the analysis of data from control and DA treatment. The Kolmogorov–Smirnoff test was used to assess the significance of the cumulative probability plots. N in the text represents the number of cells examined.

Chemicals

SCH23390, LE300, SKF38393, SKF81297, sulpiride, corynanthine, mibefradil, ZD7288, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), thapsigargin, doxazosin, and TTX were purchased from Tocris Cookson Inc. (Ellisville, MO, USA). Other chemical reagents were Sigma-Aldrich Products. All the drugs were initially prepared as a stock solution that was frozen below −20°C till use. The stock solution was diluted in the extracellular solution to make the final concentrations applied to the slices. When dimethyl sulfoxide (DMSO) or other vehicles were required to dissolve drugs, the final concentration of the vehicles was kept <0.1%. For experiments involving inhibitors, slices were usually pretreated with the extracellular solution containing the inhibitors for at least 20 min and the same concentration of the drugs were continuously applied in the bath to ensure a complete inhibition of the targets.

Results

DA Increases the Frequency Not the Amplitude of sIPSCs Recorded From Entorhinal Neurons via Activation of α1 Adrenergic Receptors

We examined the effects of DA on GABAA receptor-mediated sIPSCs recorded from the principal neurons in each layer of the EC. Stellate and pyramidal neurons are the principal neurons in layer II, whereas pyramidal neurons are the major neuronal type in layers III and V. In layer II stellate neurons, application of DA (100 µM) for 8 min significantly increased the frequency of sIPSCs to 198 ± 18% of control (n = 13, P < 0.001, Fig. 1AC) without altering the amplitude of sIPSCs significantly (103 ± 4% of control, n = 13, P = 0.97, Fig. 1A,D). DA concentration dependently increased the frequency of sIPSCs (effective concentration range: 3–100 µM) with an EC50 value of 3.6 μM (Fig. 1E). Similarly, application of DA significantly increased the frequency (F) with no effects on the amplitude (A) of sIPSCs recorded from the pyramidal neurons in layer II (F: 227 ± 20% of control, n = 5, P = 0.003; A: 114 ± 13% of control, n = 5, P = 0.35, Fig. 1F), layer III (F: 189 ± 23% of control, n = 5, P = 0.02; A: 113 ± 12% of control, n = 5, P = 0.34, Fig. 1F), and layer V (F: 229 ± 20% of control, n = 5, P = 0.003; A: 109 ± 6% of control, n = 5, P = 0.22, Fig. 1F). Whereas these results indicate that DA facilitates the frequency of sIPSCs in all the principal neurons in the EC, we used layer II stellate neurons as an example to determine the underlying cellular and molecular mechanisms for the rest of the experiments.

Figure 1.

DA increases the frequency but not the amplitude of sIPSCs recorded from entorhinal neurons. (A) sIPSCs recorded from a layer II stellate neuron before, during, and after the application of DA (100 μM). (B) Time course of the sIPSC frequency averaged from 13 stellate neurons. (C) Cumulative frequency distribution from a layer II stellate neuron before, during, and after the application of DA. (D) Cumulative amplitude distribution from the same cell before, during, and after the application of DA. The flat line part of the curves was generated because zero events were detected at the amplitudes below threshold. (E) Concentration–response curve of DA. Numbers in the parenthesis are the numbers of cells recorded. (F) Bath application of DA (100 μM) significantly enhanced the frequency (F) with no effects on the amplitude (A) of sIPSCs recorded from the pyramidal neurons in layer II (L2), layer III (L3), and layer V (L5). *P < 0.05, **P < 0.01.

Figure 1.

DA increases the frequency but not the amplitude of sIPSCs recorded from entorhinal neurons. (A) sIPSCs recorded from a layer II stellate neuron before, during, and after the application of DA (100 μM). (B) Time course of the sIPSC frequency averaged from 13 stellate neurons. (C) Cumulative frequency distribution from a layer II stellate neuron before, during, and after the application of DA. (D) Cumulative amplitude distribution from the same cell before, during, and after the application of DA. The flat line part of the curves was generated because zero events were detected at the amplitudes below threshold. (E) Concentration–response curve of DA. Numbers in the parenthesis are the numbers of cells recorded. (F) Bath application of DA (100 μM) significantly enhanced the frequency (F) with no effects on the amplitude (A) of sIPSCs recorded from the pyramidal neurons in layer II (L2), layer III (L3), and layer V (L5). *P < 0.05, **P < 0.01.

We probed the involvement of DA receptors. Slices were pretreated with the selective D1-like receptor antagonist, SCH23390 (10 μM), and the extracellular solution continued to contain the same concentration of SCH23390. Under these circumstances, bath application of DA for 8 min failed to increase, but slightly and significantly decreased the frequency (80 ± 6% of control, n = 8, P = 0.01, Fig. 2A) and amplitude (82 ± 4% of control, n = 8, P = 0.004, data not shown) of sIPSCs. We further tested the effects of another D1 antagonist of distinct structure and higher potency, LE300. Application of LE300 (100 nM, Ki = 1.9 nM for D1 receptors, Kassack et al. 2002) failed to block DA-mediated augmentation of the frequency of sIPSCs (187 ± 32% of control, n = 9, P = 0.025, Fig. 2B). Because of the distinct effects of these 2 D1 antagonists, we further tested the roles of D1-like receptors by using the agonists selective for D1-like receptors. Bath application of SKF38393 (20 µM), a selective D1-like receptor agonist, failed to significantly alter the frequency (99 ± 6% of control, n = 5, P = 0.87, Fig. 2C) and the amplitude (88 ± 5% of control, n = 5, P = 0.11) of sIPSCs. Similarly, bath application of SKF81297 (20 µM), another selective D1-like receptor agonist, did not significantly alter the frequency (102 ± 4% of control, n = 6, P = 0.64, Fig. 2D) and the amplitude (87 ± 8% of control, n = 6, P = 0.14) of sIPSCs. These results suggest that activation of D1-like receptors does not increase the frequency of sIPSCs. The effect of SCH23390 may thus not be mediated by blockade of D1-like receptors but due to its inhibition on Kirs, which were required for the effects of DA (see below) because SCH23390 is also a blocker of Kirs (Kuzhikandathil and Oxford 2002; Shankar et al. 2004; Sosulina et al. 2008; Chee et al. 2011). We then tested the roles of D2-like receptors by applying the selective D2-like receptor antagonist, sulpiride. In the presence of sulpiride (100 μM), application of DA still significantly increased the frequency (207 ± 4% of control, n = 7, P = 0.02, Fig. 2E) but failed to alter the amplitude (96 ± 7% of control, n = 7, P = 0.65) of sIPSCs, suggesting that D2-like receptors are not involved. We further tested whether the facilitatory effect of DA on sIPSC frequency requires both D1- and D2-like receptors. Bath application of SKF38393 (20 µM, D1-like agonist) and quinpirole (20 µM, D2-like agonist) still failed to increase the frequency (108 ± 6% of control, n = 5, P = 0.27, Fig. 2F) and amplitude of (93 ± 4% of control, n = 5, P = 0.19) sIPSCs. These unexpected results suggest that DA receptors are not required for DA-induced enhancement of sIPSC frequency.

Figure 2.

DA facilitates sIPSC frequency via the activation of α1 adrenoreceptors, but not DA receptors. (A) Pretreatment of slices with and continuous bath application of the D1-like receptor antagonist, SCH23390 (10 μM), blocked DA-induced facilitation of sIPSC frequency. (B) Application of another D1-like receptor antagonist, LE300 (100 nM), in the same fashion failed to block DA-mediated enhancement of sIPSC frequency. (C) Bath application of the selective D1-like receptor agonist, SKF38393 (20 μM), did not increase sIPSC frequency. (D) Bath application of SKF81297 (20 μM), another selective D1-like receptor agonist, failed to facilitate the frequency of sIPSCs. (E) Application the D2-like receptor antagonist, sulpiride (100 μM), failed to alter DA-induced facilitation of sIPSC frequency significantly. (F) Bath application of the D1- and D2-like receptors agonists did not enhance the frequency of sIPSCs. (G) Application of the selective α1 antagonist, corynanthine (100 μM), blocked DA-induced enhancement of sIPSC frequency. (H) Application of another α1 antagonist, doxazosin (25 μM), failed to block DA-induced increases in sIPSC frequency at 10, 30, and 100 μM and in the presence of the dopamine-β-hydroxylase inhibitor, fusaric acid (100 μM), DA still increased sIPSC frequency. **P < 0.01, N.S., no significance.

Figure 2.

DA facilitates sIPSC frequency via the activation of α1 adrenoreceptors, but not DA receptors. (A) Pretreatment of slices with and continuous bath application of the D1-like receptor antagonist, SCH23390 (10 μM), blocked DA-induced facilitation of sIPSC frequency. (B) Application of another D1-like receptor antagonist, LE300 (100 nM), in the same fashion failed to block DA-mediated enhancement of sIPSC frequency. (C) Bath application of the selective D1-like receptor agonist, SKF38393 (20 μM), did not increase sIPSC frequency. (D) Bath application of SKF81297 (20 μM), another selective D1-like receptor agonist, failed to facilitate the frequency of sIPSCs. (E) Application the D2-like receptor antagonist, sulpiride (100 μM), failed to alter DA-induced facilitation of sIPSC frequency significantly. (F) Bath application of the D1- and D2-like receptors agonists did not enhance the frequency of sIPSCs. (G) Application of the selective α1 antagonist, corynanthine (100 μM), blocked DA-induced enhancement of sIPSC frequency. (H) Application of another α1 antagonist, doxazosin (25 μM), failed to block DA-induced increases in sIPSC frequency at 10, 30, and 100 μM and in the presence of the dopamine-β-hydroxylase inhibitor, fusaric acid (100 μM), DA still increased sIPSC frequency. **P < 0.01, N.S., no significance.

There is accumulating evidence indicating that DA can also act via the activation of α1 adrenergic receptors (Leedham and Pennefather 1986; Rey et al. 2001; Cornil et al. 2002; Zhang et al. 2004; Lazou et al. 2006; Lin et al. 2008) and α1 adrenoreceptors enhance GABAergic transmission in the EC (Lei et al. 2007). We therefore tested the hypothesis that DA increases sIPSC frequency via the activation of α1 receptors in the EC. In the presence of the selective α1 receptor antagonist, corynanthine (100 µM), application of DA (100 µM) failed to increase either the frequency (111 ± 9% of control, n = 7, P = 0.26, Fig. 2G) or the amplitude (94 ± 2% of control, n = 7, P = 0.311) of sIPSCs. We also used another α1 receptor antagonist, doxazosin, which is distinct in structure compared with corynanthine. Pretreatment of slices with and continuous bath application of doxazosin (25 µM) blocked the increase of sIPSC frequency induced by DA at 10 (93 ± 8% of control, n = 5, P = 0.45, Fig. 2H), 30 (109 ± 6% of control, n = 8, P = 0.21, Fig. 2H), and 100 µM (111 ± 7% of control, n = 9, P = 0.16, Fig. 2H). These results suggest that DA facilitates sIPSC frequency not by the activation of dopaminergic receptors, but instead by the activation of α1 adrenoreceptors.

DA could activate α1 adrenoreceptors directly or indirectly by transformation into norepinephrine within the slices via DA-β-hydroxylase. The generated norepinephrine could then bind to α1 adrenoreceptors to mediate the effects of DA. We therefore tested this possibility by applying fusaric acid, a DA-β-hydroxylase inhibitor (Nagatsu et al. 1970; Hidaka 1971). Slices were pretreated with fusaric acid (100 µM) and the same concentration of fusaric acid was continuously applied in the bath. Under these circumstances, application of DA induced a comparable enhancement of sIPSC frequency (190 ± 22% of control, n = 4, P = 0.81 vs. DA alone, Fig. 2H), suggesting that it is unlikely that the effects of DA were mediated by conversion to norepinephrine.

Endogenously Released DA Also Increases sIPSC Frequency Via Activation of α1 Receptors

We next probed the roles of endogenously released DA in modulating GABAergic transmission. Because the EC expresses DA transporter (DAT; Erickson et al. 1998), we initially bath applied DAT inhibitor to elevate synaptic DA concentration. Bath application of the selective DAT inhibitor, GBR 12935 (5 μM), failed to significantly increase the frequency of sIPSCs (91 ± 2% of control, n = 8), compared with vehicle application (91 ± 4% of control, n = 8, P = 0.95, Fig. 3A,F). One possible explanation for the negative result is that there was no tonic spontaneous DA release at the dopaminergic terminals in the EC. We therefore used an alternative approach to stimulate DA release. Amphetamine (AMPH) is a drug that increases transporter-mediated DA release (Leviel 2011). Application of AMPH (100 μM) for 8 min significantly increased the frequency of sIPSCs (191 ± 29% of control, n = 8; P = 0.02, Fig. 3B,F). To test the involvement of DAT, we applied AMPH together with the DAT inhibitor. In the presence of GBR 12935 (5 μM), application of AMPH induced a significantly smaller increase in the frequency of sIPSCs (108 ± 5% of control, n = 8), compared with the effect of the application of AMPH alone (P = 0.007, Fig. 3C,F). We further validated the involvement of α1 receptors. Application of AMPH for 8 min in the presence of doxazosin (25 μM) failed to increase sIPSC frequency significantly (108 ± 8% of control, n = 6, P = 0.35, Fig. 3D). Because AMPH has been reported to increase the releases of both DA and norepinephrine (Rothman et al. 2001; Smith and Greene 2012), we tested whether norepinephrine transporter was involved in AMPH-induced increases in sIPSC frequency. Application of talopram (1 μM), a selective blocker for the norepinephrine transporter (McConathy et al. 2004), failed to alter AMPH-induced increases in sIPSC frequency (175 ± 22.3% of control, n = 10, P = 0.7 vs. AMPH alone, Fig. 3E,F). These data together demonstrate that endogenously released DA also facilitates sIPSC frequency via the activation of α1 receptors in the EC.

Figure 3.

Endogenously released DA enhances sIPSC frequency via the activation of α1 receptors. (A) Bath application of the DAT inhibitor, GBR 12935 (5 μM), had no significant effect on sIPSC frequency compared with that of vehicle (0.1% DMSO). (B) Bath application of AMPH (100 μM) significantly increased the frequency of sIPSCs. (C) In the presence of GBR 12935, bath application of AMPH (100 μM) induced a significantly smaller increase in sIPSC frequency. (D) AMPH-mediated increase in sIPSC frequency was blocked by α1 receptor antagonist, doxazosin (25 μM). (E) Application of talopram (1 μM) failed to alter AMPH-induced enhancement of sIPSC frequency. (F) Summary bar graph. n.s., no significant difference; **P < 0.01 compared with AMPH alone.

Figure 3.

Endogenously released DA enhances sIPSC frequency via the activation of α1 receptors. (A) Bath application of the DAT inhibitor, GBR 12935 (5 μM), had no significant effect on sIPSC frequency compared with that of vehicle (0.1% DMSO). (B) Bath application of AMPH (100 μM) significantly increased the frequency of sIPSCs. (C) In the presence of GBR 12935, bath application of AMPH (100 μM) induced a significantly smaller increase in sIPSC frequency. (D) AMPH-mediated increase in sIPSC frequency was blocked by α1 receptor antagonist, doxazosin (25 μM). (E) Application of talopram (1 μM) failed to alter AMPH-induced enhancement of sIPSC frequency. (F) Summary bar graph. n.s., no significant difference; **P < 0.01 compared with AMPH alone.

DA Enhances the Frequency of mIPSCs, But Slightly Reduces the Amplitude of eIPSCs

sIPSCs represent events caused by both AP-dependent and -independent release of GABA. In contrast, mIPSCs recorded in the presence of TTX should be independent of APs. We therefore recorded mIPSCs in the presence of TTX (0.5 µM). Application of DA significantly increased the frequency (135 ± 6% of control, n = 6, P = 0.003, Fig. 4AC) without affecting the amplitude (99 ± 4% of control, n = 6, P = 0.89, Fig. 4A,D) of mIPSCs. These results suggest that DA augments presynaptic GABA release without modulating postsynaptic GABAA receptors. We also examined the effects of DA on the GABAA receptor-mediated inhibitory post synaptic current (IPSC) recorded from stellate neurons evoked by placing a stimulation electrode in a location of approximately 200 μm from the recorded neurons. We used a protocol of paired stimulation (interval: 50 ms and frequency: 0.1 Hz) to measure the paired-pulse ratio (PPR) simultaneously. Bath application of DA slightly but significantly reduced the amplitude of eIPSCs evoked by the first stimulation (87 ± 4% of control, n = 6, P = 0.023, Fig. 4E). DA-induced depression of eIPSC amplitude was presynaptic in origin, because DA significantly increased the PPR (n = 6, P = 0.002, Fig. 4F). Two potential mechanisms could be proposed to explain the discrepancy of the results regarding sIPSCs and eIPSCs. The first explanation for DA-induced depression of eIPSCs is that DA-induced enhancement of spontaneous GABA release depletes the readily releasable vesicle pool that is subsequently available for eIPSCs as suggested for 5-HT3 (Cui et al. 2012) and muscarinic (Xiao, Deng, Yang, et al. 2009) receptors. Alternatively, DA-mediated depression of eIPSCs could be due to its inhibitory effect on the AP amplitude of GABAergic interneurons, because DA-induced membrane depolarization could inactivate Na+ channels resulting in APs of lower amplitude (see below). Because sIPSCs, mIPSCs, and eIPSCs represent distinct modes of GABAergic transmission, the diverse effects of DA on these IPSCs suggest that DA exerts different actions depending on the status of the neural circuitry. The results that DA increased the frequency of sIPSCs and mIPSCs suggest that DA facilitates GABA release. We therefore further determined the mechanisms underlying DA-induced GABA release.

Figure 4.

DA augments the frequency with no effects on the amplitude of mIPSCs recorded in the presence of TTX, but attenuates the amplitude of eIPSC. (A) mIPSC current traces recorded from a stellate neuron before, during, and after the application of DA. (B) Time course of mIPSC frequency summarized from 6 stellate neurons. (C) Cumulative frequency distribution of mIPSCs before, during, and after the application of DA. Note that DA reduced the interval of mIPSCs suggesting an increase in mIPSC frequency. (D) Cumulative amplitude distribution of mIPSCs before, during, and after the application of DA. Note that DA did not change the amplitude of mIPSCs. The flat line part of the curves was generated because zero events were detected at the amplitudes below threshold. (E) DA depressed the amplitude of eIPSCs recorded from layer II stellate neurons by application of a protocol comprising paired stimulation (50 ms interval at 0.1 Hz). The amplitudes of the eIPSCs evoked by the first stimulation were normalized to the average of the 5 min before application of DA. Upper panel shows the average of 6 eIPSCs before and during the application of DA. (F) DA increased the PPR. Upper panel shows the eIPSCs before and during the application of DA scaled to the amplitude evoked by the first stimulation. Note that the amplitude of the second eIPSC in the presence of DA is larger than control.

Figure 4.

DA augments the frequency with no effects on the amplitude of mIPSCs recorded in the presence of TTX, but attenuates the amplitude of eIPSC. (A) mIPSC current traces recorded from a stellate neuron before, during, and after the application of DA. (B) Time course of mIPSC frequency summarized from 6 stellate neurons. (C) Cumulative frequency distribution of mIPSCs before, during, and after the application of DA. Note that DA reduced the interval of mIPSCs suggesting an increase in mIPSC frequency. (D) Cumulative amplitude distribution of mIPSCs before, during, and after the application of DA. Note that DA did not change the amplitude of mIPSCs. The flat line part of the curves was generated because zero events were detected at the amplitudes below threshold. (E) DA depressed the amplitude of eIPSCs recorded from layer II stellate neurons by application of a protocol comprising paired stimulation (50 ms interval at 0.1 Hz). The amplitudes of the eIPSCs evoked by the first stimulation were normalized to the average of the 5 min before application of DA. Upper panel shows the average of 6 eIPSCs before and during the application of DA. (F) DA increased the PPR. Upper panel shows the eIPSCs before and during the application of DA scaled to the amplitude evoked by the first stimulation. Note that the amplitude of the second eIPSC in the presence of DA is larger than control.

Because mIPSCs recorded in the presence of TTX are AP-independent, our results that DA facilitated the frequency of mIPSCs recorded in the presence of TTX suggest that there should be at least an AP-independent mechanism involved. We therefore tested whether extracellular Ca2+ was required for the effects of DA on GABAergic transmission by recording sIPSCs and mIPSCs from layer II stellate neurons in a Ca2+-free extracellular solution. The extracellular Ca2+ was replaced by the same concentration of Mg2+, and 1 mM of EGTA was included in the extracellular solution to chelate the ambient residual Ca2+. Under these circumstances, bath application of DA failed to increase the frequencies of sIPSCs (n = 11, P = 0.42, Fig. 5A) and mIPSCs (n = 6, P = 0.94, Fig. 5B). These data together suggest that extracellular Ca2+ is required for the effects of DA on GABAergic transmission.

Figure 5.

Ca2+ influx via T-type Ca2+ channels is required for DA-induced facilitation of GABAergic transmission. (A and B) Depletion of extracellular Ca2+ by replacing extracellular Ca2+ with Mg2+ and inclusion of 1 mM EGTA in the extracellular solution prevented DA-induced enhancement of sIPSC (A) and mIPSC (B) frequency. (C and D) Bath application of the high-threshold voltage-gated Ca2+ channel blocker, Cd2+ (100 μM), failed to block DA-induced enhancement of sIPSC (C) and mIPSC (D) frequency. (E and F) Bath application of the low-threshold T-type Ca2+ channel blocker, Ni2+ (200 μM), significantly reduced DA-induced augmentation of sIPSC frequency (E) and blocked DA-mediated increment of mIPSC frequency (F). (G and H) Bath application of the T-type Ca2+ channel blocker, mibefradil (15 μM), significantly reduced DA-induced increase of sIPSC frequency (G) and blocked DA-mediated enhancement of mIPSC frequency (H).

Figure 5.

Ca2+ influx via T-type Ca2+ channels is required for DA-induced facilitation of GABAergic transmission. (A and B) Depletion of extracellular Ca2+ by replacing extracellular Ca2+ with Mg2+ and inclusion of 1 mM EGTA in the extracellular solution prevented DA-induced enhancement of sIPSC (A) and mIPSC (B) frequency. (C and D) Bath application of the high-threshold voltage-gated Ca2+ channel blocker, Cd2+ (100 μM), failed to block DA-induced enhancement of sIPSC (C) and mIPSC (D) frequency. (E and F) Bath application of the low-threshold T-type Ca2+ channel blocker, Ni2+ (200 μM), significantly reduced DA-induced augmentation of sIPSC frequency (E) and blocked DA-mediated increment of mIPSC frequency (F). (G and H) Bath application of the T-type Ca2+ channel blocker, mibefradil (15 μM), significantly reduced DA-induced increase of sIPSC frequency (G) and blocked DA-mediated enhancement of mIPSC frequency (H).

Since extracellular Ca2+ is required for DA's presynaptic actions, we also tested whether Ca2+ influx via voltage-gated Ca2+ channels is necessary for DA-mediated increases in the frequencies of sIPSCs and mIPSCs. Inclusion of CdCl2 (100 µM), a blocker for high-threshold voltage-dependent Ca2+ channels, in the extracellular solution, failed to block DA-induced increases the frequency of sIPSCs (n = 5, P = 0.67 vs. control, Fig. 5C) and mIPSCs (n = 5, P = 0.004, Fig. 5D). However, addition of NiCl2 (200 µM), a blocker for the low-threshold T-type Ca2+ channels, significantly reduced DA-induced increases in the frequency of sIPSCs (n = 14, P = 0.033 vs. control, Fig. 5E). DA-mediated augmentation of mIPSC frequency was blocked in the extracellular solution containing Ni2+ (n = 5, P = 0.96, Fig. 5F). We further examined the involvement of T-type Ca2+ channels with mibefradil, another T-type Ca2+ channels blocker. Bath application of mibefradil (15 µM) significantly reduced DA-induced increases in sIPSC frequency (113 ± 5% of control, n = 8, P = 0.04 vs. DA alone, Fig. 5G) but blocked completely DA-mediated facilitation of mIPSC frequency (106 ± 11% of control, n = 5, P = 0.64 vs. baseline, Fig. 5H). These data together indicate that T-type Ca2+ channels are required for DA-induced facilitation of GABAergic transmission.

DA Depolarizes GABAergic Interneurons in the EC

Because our results point to a presynaptic mechanism underlying DA-induced facilitation of GABA release, we also examined the effects of DA on GABAergic interneurons by recording from the interneurons in layer III of the EC. As demonstrated previously (Deng and Lei 2008), interneurons in the EC can be divided into 2 types according to their electrophysiological properties. Type I interneurons showed little voltage sag in response to hyperpolarizing current injection and no rebound burst firing (Fig. 6A1), whereas Type II interneurons displayed prominent voltage sag in response to hyperpolarizing current injection and rebound burst firing (Fig. 6B1). After having electrophysiologically identified the interneurons, we recorded the RMPs by washing with TTX (0.5 µM) in the extracellular solution. Because application of DA-induced depolarization in both types of interneurons (Fig. 6A2,B2), the data recorded from Type I and Type II interneurons were pooled. Application of DA depolarized the interneurons recorded at the RMPs (control: −66.2 ± 0.8 mV; DA: −62.0 ± 1.0 mV, n = 9, P < 0.001; Fig. 6C, left) and slightly but significantly increased the input resistance of the interneurons (control: 304 ± 54 MΩ; DA: 330 ± 55 MΩ, n = 9, P = 0.047; Fig. 6C, right). Furthermore, application of DA induced a small inward HC recorded at −60 mV from interneurons (−7.5 ± 1.9 pA, n = 5, P = 0.018, Fig. 6D). We also probed the roles of DA in AP firing by including in the extracellular solution containing (in µM) 10 DNQX, 50 APV, 10 bicuculline, and 2 CGP55845 to block glutamatergic and GABAergic transmission. Because the RMPs of the interneurons were usually negative to −60 mV, interneurons did not show spontaneous firing at their RMPs. The depolarization generated by DA was not large enough to raise the RMPs to the threshold for AP firing. We therefore injected positive currents to elevate the membrane potential to just above the threshold to induce AP firing. Under these circumstances, application of DA significantly increased the firing frequency of APs (n = 7, P = 0.036, Fig. 6E1,E2), but slightly decreased the AP amplitude (89 ± 1% of control, n = 7, P < 0.001, Fig. 6E1). DA-induced depression of AP amplitude might be due to its depolarizing effect resulting in inactivation of Na+ channels.

Figure 6.

DA depolarizes GABAergic interneurons in the EC. (A1 and A2) Bath application of DA generated membrane depolarization and increased the input resistance of Type I interneurons in the EC. (A1) Voltage changes in response to current injection (±150 pA) in a Type I interneuron. (A2) Application of DA (100 μM) generated membrane depolarization and increased input resistance in the same interneuron. RMP was recorded in the current-clamp mode and a hyperpolarizing current (−50 pA, 500 ms) was injected every 20 s to measure the input resistance. Note that DA generated depolarization and increased the input resistance. To exclude the influence of DA-induced membrane depolarization on the input resistance, a negative current (−8 pA indicated by the horizontal bar) was injected briefly to bring the membrane potential back to the initial level. Under these conditions, the voltage responses induced by the injection of hyperpolarizing currents (−50 pA, 500 ms) were still larger compared with control, suggesting that DA-induced increases in input resistance are not secondary to its effect on membrane depolarization. Inset is the voltage traces taken before (a) and during (b) the application of DA when the negative current was injected. (B1 and B2) Bath application of DA generated membrane depolarization and increased the input resistance of Type II interneurons in the EC. The experiment was performed in the same fashion as Type I interneurons. (C) Pooled data for DA induced depolarization (left) and increase in input resistance (right). Empty circles represent values from individual cells and solid symbols denote the average values. (D) Application of DA induced an inward HC in interneurons (n = 5). (E1 and E2) Bath application of DA increased AP firing frequency in interneurons. APs were evoked by injecting a positive current to elevate the membrane potential just above the threshold for firing. (E1) APs recorded from an interneuron before, during, and after the application of DA. (E2) Pooled time course of AP firing (n = 7).

Figure 6.

DA depolarizes GABAergic interneurons in the EC. (A1 and A2) Bath application of DA generated membrane depolarization and increased the input resistance of Type I interneurons in the EC. (A1) Voltage changes in response to current injection (±150 pA) in a Type I interneuron. (A2) Application of DA (100 μM) generated membrane depolarization and increased input resistance in the same interneuron. RMP was recorded in the current-clamp mode and a hyperpolarizing current (−50 pA, 500 ms) was injected every 20 s to measure the input resistance. Note that DA generated depolarization and increased the input resistance. To exclude the influence of DA-induced membrane depolarization on the input resistance, a negative current (−8 pA indicated by the horizontal bar) was injected briefly to bring the membrane potential back to the initial level. Under these conditions, the voltage responses induced by the injection of hyperpolarizing currents (−50 pA, 500 ms) were still larger compared with control, suggesting that DA-induced increases in input resistance are not secondary to its effect on membrane depolarization. Inset is the voltage traces taken before (a) and during (b) the application of DA when the negative current was injected. (B1 and B2) Bath application of DA generated membrane depolarization and increased the input resistance of Type II interneurons in the EC. The experiment was performed in the same fashion as Type I interneurons. (C) Pooled data for DA induced depolarization (left) and increase in input resistance (right). Empty circles represent values from individual cells and solid symbols denote the average values. (D) Application of DA induced an inward HC in interneurons (n = 5). (E1 and E2) Bath application of DA increased AP firing frequency in interneurons. APs were evoked by injecting a positive current to elevate the membrane potential just above the threshold for firing. (E1) APs recorded from an interneuron before, during, and after the application of DA. (E2) Pooled time course of AP firing (n = 7).

Ionic Mechanisms Underlying DA-Induced Depolarization of Interneurons

We recorded the RMPs of the interneurons to further determine the underlying ionic mechanisms. DA has been shown to facilitate the hyperpolarization-activated channels (Ih channels) in layer V pyramidal neurons (Rosenkranz and Johnston 2006). We therefore examined whether Ih channels are involved in DA-induced depolarization. Extracellular application of the selective Ih channel blocker, ZD7288 (20 µM), failed to block DA-induced depolarization (n = 5, P = 0.7 vs. DA alone, Fig. 7A,G), suggesting that DA-mediated membrane depolarization of interneurons is not dependent on Ih channels. If DA-induced membrane depolarization of interneurons is due to the opening of cation channels, the influx of extracellular Na+ should be the major ions to mediate membrane depolarization. However, replacement of extracellular NaCl with the same concentration of NMDG-Cl failed to alter DA-induced depolarization (n = 5, P = 0.47 vs. DA alone, Fig. 7B,G). Because extracellular Ca2+ is required for DA-induced increases in the frequencies of sIPSCs and mIPSCs, we also tested whether extracellular Ca2+ is required for DA-induced depolarization of interneurons. Replacement of extracellular Ca2+ with the same concentration of Mg2+ and simultaneous inclusion of 1 mM EGTA in the extracellular solution did not significantly change DA-induced depolarization (n = 7, P = 0.41 vs. DA alone, Fig. 7C,G). These data together suggest that DA does not depolarize interneurons by opening a cationic conductance. Furthermore, bath application of NiCl2 (200 µM) failed to significantly change DA-induced depolarization (n = 6, P = 0.82 vs. DA alone, Fig. 7D,G), suggesting that T-type Ca2+ channels are not involved in DA-mediated depolarization of the interneurons. Finally, we determined the requirement for intracellular Ca2+ in mediating the postsynaptic DA response. DA induced a smaller level of depolarization (n = 6, P = 0.005 vs. DA alone, Fig. 7E,G) when BAPTA (10 mM) was included in the recording pipettes. However, inclusion of thapsigargin (10 µM) in the pipettes failed to alter DA-induced depolarization significantly (n = 5, P = 0.69 vs. DA alone, Fig. 7F,G), suggesting that intracellular Ca2+ release is not required for DA-induced depolarization. One explanation for the result that intracellular application of BAPTA significantly reduced DA-induced depolarization is that the effects of DA may require the functions of some Ca2+-dependent signals.

Figure 7.

DA-induced depolarization of interneurons does not require the function of Ih channels and is independent of extracellular Na+ and Ca2+, but is affected by intracellular Ca2+ concentration. (A) Bath application of the Ih channel blocker, ZD7288 (20 μM), did not block DA-induced depolarization. (B) Replacement of extracellular NaCl with NMDG-Cl did not alter DA-induced depolarization. (C) Substitution of extracellular Ca2+ with Mg2+ and inclusion of EGTA (1 mM) in the extracellular solution failed to change DA-induced depolarization. (D) Inclusion of Ni2+ (200 μM) in the extracellular solution did not block DA-induced depolarization. (E) Inclusion of BAPTA (10 mM) in the recording pipettes reduced DA-induced depolarization, suggesting that intracellular Ca2+ concentration is related to DA-induced depolarization possibly by affecting Ca2+-dependent intracellular signals. (F) Intracellular application of thapsigargin (10 μM) via the recording pipettes failed to modify DA-mediated depolarization, suggesting that intracellular Ca2+ release is not required for DA-mediated depolarization. (G) Pooled data.

Figure 7.

DA-induced depolarization of interneurons does not require the function of Ih channels and is independent of extracellular Na+ and Ca2+, but is affected by intracellular Ca2+ concentration. (A) Bath application of the Ih channel blocker, ZD7288 (20 μM), did not block DA-induced depolarization. (B) Replacement of extracellular NaCl with NMDG-Cl did not alter DA-induced depolarization. (C) Substitution of extracellular Ca2+ with Mg2+ and inclusion of EGTA (1 mM) in the extracellular solution failed to change DA-induced depolarization. (D) Inclusion of Ni2+ (200 μM) in the extracellular solution did not block DA-induced depolarization. (E) Inclusion of BAPTA (10 mM) in the recording pipettes reduced DA-induced depolarization, suggesting that intracellular Ca2+ concentration is related to DA-induced depolarization possibly by affecting Ca2+-dependent intracellular signals. (F) Intracellular application of thapsigargin (10 μM) via the recording pipettes failed to modify DA-mediated depolarization, suggesting that intracellular Ca2+ release is not required for DA-mediated depolarization. (G) Pooled data.

DA can inhibit background K+ channels to generate membrane depolarization. Our result that DA increased the input resistance also supports the involvement of K+ channels. To test the roles of K+ channels, we first replaced the intracellular K+-gluconate with NMDG-gluconate and recorded the changes of membrane potentials in response to DA. Under these circumstances, bath application of DA failed to induce membrane depolarization (n = 5, P = 0.31, Fig. 8A,K). Secondly, we used a ramp protocol to measure the reversal potential of the current induced by DA. The DA-induced current had a reversal potential of −85.6 ± 6.5 mV (n = 5, Fig. 8B,C), which was close to the calculated K+ reversal potential (−85.4 mV). These data indicate the involvement of K+ channels. We also noticed that the current generated by DA showed an inward rectification (Fig. 8C), suggesting that DA inhibits Kirs. Consistent with this, inclusion of Ba2+ (1 mM) in the extracellular solution blocked DA-induced depolarization (n = 6, P = 0.78 vs. baseline, Fig. 8D,K), further supporting the involvement of Kirs. Moreover, bath application of Ba2+ alone significantly increased the frequency of sIPSCs (n = 3, P = 0.004, Fig. 8E). In the presence of Ba2+, application of DA did not increase but slightly reduced the frequency of (n = 3, P = 0.015, Fig. 8E) sIPSC. Similarly, Ba2+ application blocked DA-induced increases in mIPSC frequency (n = 6, P = 0.72, Fig. 8F). We also tested the effects of SCH23390 on DA-induced depolarization. Slices were pretreated with SCH23390 (10 µM) and the extracellular solution was continuously perfused with the same concentration of SCH23390. Application of SCH23390 prevented DA-induced depolarization (n = 6, P = 0.97, Fig. 8G,K). However, application of the selective D1-like agonist, SKF38393 (40 µM), failed to depolarize interneurons (n = 6, P = 0.27 vs. baseline, Fig. 8H,K), but subsequent application of DA still induced depolarization in the same neurons (Fig. 8H). These results suggest that the blocking effect of SCH23390 was not mediated by antagonizing D1-like receptors, but by blockade of Kirs. We further tested the roles of α1 receptors in DA-induced depolarization of interneurons. Application of the α1 receptor antagonist, corynanthine (100 µM), blocked DA-induced depolarization (n = 5, P = 0.41 vs. baseline, Fig. 8I,K) and application of the selective α1 receptor agonist, phenylephrine (100 µM), induced depolarization of interneurons (n = 6, P = 0.002, Fig. 8J,K) demonstrating the involvement of α1 receptors.

Figure 8.

DA-induced depolarization of interneurons is mediated by inhibition of Kirs. (A) DA did not induce conspicuous depolarization when the intracellular K+ was replaced with NMDG. (B) Current–voltage relationship recorded by a ramp protocol (from −110 to −50 mV) in the extracellular solution containing 3.5 mM K+ before and during the application of DA. Traces in the figure were averaged traces from 5 cells. (C) The DA-generated net current obtained by subtraction of the control from that in the presence of DA has a reversal potential at approximately −85.6 mV close to the calculated K+ reversal potential (∼ −85.4 mV). Note that the DA-sensitive current showed an inward rectification. (D) Bath application of Ba2+ blocked DA-induced depolarization. (E) Bath application of Ba2+ increased sIPSC frequency and subsequent application of DA slightly reduced sIPSC frequency. (F) Bath application of Ba2+ increased mIPSC frequency and blocked DA-induced increases in mIPSC frequency. (G) Pretreatment of slices with and continuous bath application of SCH23390 blocked DA-induced depolarization. (H) Bath application of SKF38393 (40 μM) did not induce depolarization, but subsequent application of DA still induced depolarization in the same cell. (I) Pretreatment of slices with and continuous bath application of corynanthine (100 μM) blocked DA-induced depolarization. (J) Bath application of the selective α1 receptor agonist, phenylephrine (100 μM), induced depolarization of an interneuron. (K) Pooled data. **P < 0.01 versus baseline.

Figure 8.

DA-induced depolarization of interneurons is mediated by inhibition of Kirs. (A) DA did not induce conspicuous depolarization when the intracellular K+ was replaced with NMDG. (B) Current–voltage relationship recorded by a ramp protocol (from −110 to −50 mV) in the extracellular solution containing 3.5 mM K+ before and during the application of DA. Traces in the figure were averaged traces from 5 cells. (C) The DA-generated net current obtained by subtraction of the control from that in the presence of DA has a reversal potential at approximately −85.6 mV close to the calculated K+ reversal potential (∼ −85.4 mV). Note that the DA-sensitive current showed an inward rectification. (D) Bath application of Ba2+ blocked DA-induced depolarization. (E) Bath application of Ba2+ increased sIPSC frequency and subsequent application of DA slightly reduced sIPSC frequency. (F) Bath application of Ba2+ increased mIPSC frequency and blocked DA-induced increases in mIPSC frequency. (G) Pretreatment of slices with and continuous bath application of SCH23390 blocked DA-induced depolarization. (H) Bath application of SKF38393 (40 μM) did not induce depolarization, but subsequent application of DA still induced depolarization in the same cell. (I) Pretreatment of slices with and continuous bath application of corynanthine (100 μM) blocked DA-induced depolarization. (J) Bath application of the selective α1 receptor agonist, phenylephrine (100 μM), induced depolarization of an interneuron. (K) Pooled data. **P < 0.01 versus baseline.

Discussion

Our results demonstrate that DA increases the frequency without affecting the amplitude of sIPSCs and mIPSCs in the EC. The effects of DA are not mediated by DA receptors, but by α1 adrenoreceptors. Endogenously released DA exerts the same effects on GABAergic transmission. DA-induced increases in the frequencies of sIPSCs and mIPSCs are due to DA-mediated depolarization of GABAergic interneurons resulting in the facilitation of AP firing frequency and the activation of T-type Ca2+ channels. DA-mediated depolarization of interneurons is caused by the inhibition of Kirs.

DA increases the frequencies of sIPSCs and mIPSCs with no effects on their amplitudes in the EC. These results indicate that DA increases presynaptic GABA release with no effects on postsynaptic GABAA receptors. Because sIPSCs are usually considered to be AP-dependent, whereas mIPSCs recorded in the presence of TTX are not, our results suggest that DA facilitates GABA release at least in part by an AP-independent mechanism. Because DA-mediated increases in the frequencies of sIPSCs and mIPSCs are dependent on extracellular Ca2+, we examined the involvement of voltage-gated Ca2+ channels. Bath application of Cd2+, a blocker of high-threshold voltage-gated Ca2+ channels, failed to block DA-mediated increases in the frequencies of sIPSC and mIPSC, whereas application of Ni2+ and mibefradil, 2 blockers of T-type Ca2+ channels, significantly reduced DA-induced facilitation of the frequency of sIPSCs and mIPSCs indicating the involvement of T-type Ca2+ channels. Furthermore, whereas DA depolarizes GABAergic interneurons, T-type Ca2+ channels are not required for DA-induced depolarization of interneurons because bath application of Ni2+ did not alter DA-mediated depolarization. Our results therefore suggest that DA depolarizes interneurons, which in turn facilitates the activity of T-type Ca2+ channels. Increased influx of Ca2+ through T-type Ca2+ channels leads to increases in GABA release.

T-type Ca2+ channels are low-voltage-activated Ca2+ channels that control Ca2+ entry in excitable cells during small depolarization above resting potentials. For example, sustained depolarization generated by elevation of extracellular K+ concentration (Barish 1991; Varnai et al. 1995; Bao et al. 1998; Boyer et al. 1998; Jensen et al. 2004) or direct neuronal depolarization (Varnai et al. 1995; Lu et al. 1997; Kawai and Miyachi 2001; Pan et al. 2001; Bessaih et al. 2008) activates T-type Ca2+ channels. Although bath application of DA increases the firing frequency of APs when the membrane potential of the interneurons is raised above threshold by injection of positive current, application of DA is incapable of inducing APs when interneurons are at rest. Under our recording conditions, interneurons rest negative to −60 mV and the average depolarization generated by DA is approximately 3–4 mV. The threshold for AP firing in the interneurons is at least positive to −50 mV. Therefore, DA makes little contribution to increasing the firing rate of the interneurons at rest. However, DA-induced, small subthreshold depolarization would likely shift the activation curve of T-type Ca2+ channels to the direction of negative potentials (Varnai et al. 1995) thereby increasing Ca2+ influx. Ca2+ influx via T-type Ca2+ channels has been shown to facilitate the release of neurotransmitters including GABA (Carbone et al. 2006).

Our results do not support a role of nonselective cation channel activation in DA-induced depolarization of interneurons. If opening of a nonselective cationic conductance is responsible for DA-induced facilitation of GABA release, the influxes of extracellular Na+ and Ca2+ should be the major cations to mediate the depolarization of interneurons. The result that substitution of extracellular NaCl with NMDG-Cl failed to alter DA-induced depolarization does not support a role for Na+ in DA-induced depolarization of interneurons. However, our results suggest a role for extracellular Ca2+ influx in DA-mediated enhancement of GABA release, because depletion of extracellular Ca2+ blocked DA-induced increases in the frequencies of sIPSCs and mIPSCs. Because exclusion of extracellular Ca2+ does not alter the DA-induced depolarization of interneurons, the effects of Ca2+ are likely secondary to DA-induced depolarization. Our results that blocking T-type Ca2+ channels reduces DA-induced increases in the frequencies of sIPSCs and mIPSCs, but does not alter DA-mediated depolarization of interneurons, indicate that the required Ca2+ is through T-type Ca2+ channels secondary to membrane depolarization.

Activation of D1-like receptors in the pyramidal neurons of the EC generates membrane hyperpolarization via the activation of Ih channels (Rosenkranz and Johnston 2006). Our results do not support a role of Ih channels in DA-induced facilitation of GABA release based on the following lines of evidence. First, at the RMP (∼ −60 mV) of the interneurons, Ih channels should be open. If Ih channels are involved, DA should increase the function of Ih channels to generate membrane depolarization. Bath application of the Ih channel blocker, ZD7288, should block DA-induced depolarization. However, bath application of DA still induced a comparable depolarization in the presence of ZD7288. Secondly, if Ih channels are involved, influx of Na+ should be responsible for depolarization. However, replacing extracellular Na+ with NMDG did not alter DA-induced depolarization. Thirdly, whereas Type II interneurons exhibit a sag response that is generated by the activation of Ih channels, Type I do not show noticeable sag, suggesting that Type I interneurons do not express Ih channels. However, DA depolarizes both Type I and Type II interneurons, suggesting that Ih channels are not responsible for DA-induced depolarization. Fourthly, if Ih channels are involved, DA-mediated activation of Ih channels should reduce the input resistance. Nevertheless, application of DA increases the input resistance further excluding the contribution of Ih channels. Therefore, we conclude that the depolarization of interneurons is largely independent of cation influx.

Our results support a role of Kirs in DA-induced depolarization of interneurons based on the following pieces of evidence. First, the reversal potential of DA-generated currents is close to the K+ reversal potential. Secondly, replacement of intracellular K+ with NMDG blocked DA-induced depolarization. These 2 lines of evidence buttress the involvement of K+ channels. Thirdly, the current–voltage relationship of the DA-induced current exhibits an inward rectification. Fourthly, bath application of Ba2+, a Kir blocker, annuls the DA-induced depolarization of interneurons and the facilitatory effects of DA on sIPSCs and mIPSCs, further supporting the participation of Kirs. Whereas the results that application of SCH23390 blocks both DA-mediated increases in sIPSC frequency and DA-mediated depolarization of interneurons could be explained either by the involvement of D1-like receptors or by SCH23390-mediated blockade of Kirs, our results support the latter. If the blocking effects of SCH23390 are mediated by blockade of D1-like receptors, application of the selective D1-like receptor agonists should also exert the same actions as DA. However, our results showed that application of D1-like receptor agonists or co-application of the agonists for D1- and D2-like receptors failed to have any effects on sIPSCs and the RMPs of the interneurons, suggesting that the blocking effects of SCH23390 are not mediated by interaction with D1-like receptors. Coincidently, we found that DA generates membrane depolarization in the entorhinal interneurons via inhibition of Kirs, which can be blocked by SCH23390 (Kuzhikandathil and Oxford 2002; Shankar et al. 2004; Sosulina et al. 2008; Chee et al. 2011). Further evidence to support the idea that the effects of SCH23390 were mediated by blocking Kirs instead of D1-like receptors is that application of a structurally distinct D1 receptor antagonist, LE300, failed to prevent DA-induced facilitation of sIPSC frequency. Moreover, the results that DA-induced facilitation of sIPSCs and mIPSCs and depolarization of interneurons are blocked by application of α1 adrenoreceptor antagonists suggest that D1-like receptors are not involved. Our results demonstrate that DA depolarizes GABAergic interneurons via α1 receptor-mediated inhibition of Kirs. Consistent with our findings, DA has been shown to inhibit Kirs (Gorelova et al. 2002; Dong et al. 2004; Witkowski et al. 2008; Govindaiah et al. 2010; Podda et al. 2010). Different from our results is that application of the D1-like receptor agonists in these studies exerts the same actions as DA, suggesting the involvement of D1-like receptors.

Whereas our results demonstrate that DA facilitates GABAergic transmission via activation of α1 adrenoreceptors, there are still differences between the effects of DA and norepinephrine, which also facilitates GABAergic transmission in the EC. First, DA increases only the frequency of sIPSCs, whereas norepinephrine facilitates both the frequency and amplitude of sIPSCs. Secondly, extracellular Ca2+ is required for DA-induced increases in the frequency of sIPSCs and mIPSCs, but not required for the effects of norepinephrine on GABA release. Thirdly, DA transiently increases the action potential firing frequency in interneurons, whereas norepinephrine has no obvious effects on the firing frequency of action potentials and holding currents recorded from the interneurons. Several mechanisms can be proposed to explain the discrepancy between the effects of DA and those of norepinephrine on GABAergic transmission. First, norepinephrine interacts with many different types of receptors including α1, α2, and β adrenoreceptors as well as D2 DA receptors (Robbins et al. 1988), whereas DA activates α1, α2, and β adrenoreceptors (Rajfer et al. 1988; Anfossi et al. 1993; Lee et al. 1998; Ouedraogo et al. 1998; Ooi and Colucci 2001; Cornil et al. 2002) in addition to activating DA receptors. Activation of these receptors likely produces distinct or even opposite effects. Whereas our previous results demonstrate a role for α1 receptors in norepinephrine-induced facilitation of GABAergic transmission in the EC, the permissive or shrouded roles of other receptors activated by norepinephrine are unknown. Secondly, there are several different subtypes of α1 adrenoreceptors. As demonstrated previously (Rey et al. 2001), norepinephrine and DA may activate distinct subtypes of α1 receptors. Thirdly, whereas DA and norepinephrine exert promiscuous effects on different receptors, there are significant differences with regard to the affinities of the receptors activated by DA and norepinephrine; for example, DA has only 1 of 50 the affinity of norepinephrine for α1 receptors (Leedham and Pennefather 1986). Receptors activated by distinct agonists of different affinities likely generate distinguishable intracellular signaling events resulting in dissimilar actions. Thirdly, although our results do not support a role for D1- and D2-like receptors in the effects of DA on GABAergic transmission, it is still possible that DA modulates GABAergic transmission by a cooperative effects on α1, D1, and D2 receptors because there is strong evidence demonstrating an interaction of α1, D1, and D2 receptors (Gioanni et al. 1998; Wadenberg et al. 2000; Stuchlik et al. 2008).

Synaptic DA concentration can reach approximately 100 µM (Ford et al. 2009). At this concentration, DA or DA receptor agonists have been reported to increases sIPSC frequency in the lateral amygdala (Loretan et al. 2004), cerebral cortex (Zhou and Hablitz 1999; Seamans et al. 2001), and thalamus (Munsch et al. 2005). In the EC, the effect of DA on GABAergic transmission was reliably observed when DA was applied at a concentration range of 3–100 µM. In the present study, we performed a series of experiments to test the role of endogenously released DA in modulating GABAergic transmission. We initially tried to elevate synaptic DA concentration by bath application of the DAT inhibitor, GBR 12935. However, bath application of the DAT inhibitor failed to increase the frequency of sIPSCs significantly. One explanation is that there is no tonic DA release at the dopaminergic projections in the EC possibly due to the severing of the terminals from their somas in our slice preparation. We also used AMPH, a drug that promotes DA efflux via interaction with the DAT (Leviel 2011). Bath application of AMPH increased sIPSC frequency and the effect of AMPH was almost completely abolished by application of GBR 12935, suggesting that the effect of AMPH is mediated by increasing endogenous DA efflux. We further demonstrate that bath application of α1 receptor antagonist blocked AMPH-induced increase in the frequency of sIPSCs, whereas application of the inhibitor for norepinephrine transporter failed to affect the effect of AMPH. These results together indicate that endogenously released DA is capable of facilitating GABA release in the EC.

In the EC, DA usually exerts an overall inhibitory effect. For example, DA has been shown to inhibit the excitability of pyramidal neurons (Rosenkranz and Johnston 2006; Mayne et al. 2013), excitatory synaptic transmission (Pralong and Jones 1993; Stenkamp et al. 1998; Behr et al. 2000; Caruana and Chapman 2008), and synaptic plasticity (Caruana et al. 2007). DA has bidirectional effects on excitatory synaptic transmission with low concentrations enhancing, and high concentrations depressing it (Caruana et al. 2006). Consistent with the generally inhibitory roles of DA in the EC, our results indicate that DA facilitates GABA release. GABAergic transmission in the EC synchronizes neural network activities and serves as the precision clockwork for gamma and theta oscillations (Cutsuridis and Hasselmo 2012). Neural oscillatory events are thought to be crucially involved in various cognitive processes. Because the functions of the EC are closely related to the processes of learning and memory (Steffenach et al. 2005), Alzheimer's disease (Hyman et al. 1984) and schizophrenia (Prasad et al. 2004), DA-mediated modulation of GABAergic transmission would likely play a role in the modification of these physiological functions and neurological diseases.

In conclusion, our results demonstrate that DA facilitates the frequency of sIPSCs and mIPSCs, indicating that DA increases GABA release in the EC. The facilitatory effects of DA are not mediated by DA receptors but via the activation of α1 adrenergic receptors. DA inhibits Kirs to generate a small depolarization of GABAergic interneurons resulting in facilitation of T-type Ca2+ channels. Our results have revealed a collaborative role of α1 adrenoreceptors, Kirs, and T-type Ca2+ channels in DA-induced augmentation of GABA release in the EC.

Funding

This work was supported by the National Institute of Mental Health (MH082881 to S.L.).

Notes

Conflict of Interest: None declared.

References

Akil
M
Lewis
DA
The dopaminergic innervation of monkey entorhinal cortex
Cereb Cortex
 , 
1993
, vol. 
3
 (pg. 
533
-
550
)
Anfossi
G
Massucco
P
Mularoni
E
Mattiello
L
Cavalot
F
Burzacca
S
Trovati
M
Effect of dopamine on adenosine 3′,5′-cyclic monophosphate levels in human platelets
Gen Pharmacol
 , 
1993
, vol. 
24
 (pg. 
435
-
438
)
Arnold
SE
Hyman
BT
Van Hoesen
GW
Damasio
AR
Some cytoarchitectural abnormalities of the entorhinal cortex in schizophrenia
Arch Gen Psychiatry
 , 
1991
, vol. 
48
 (pg. 
625
-
632
)
Avoli
M
D'Antuono
M
Louvel
J
Kohling
R
Biagini
G
Pumain
R
D'Arcangelo
G
Tancredi
V
Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro
Prog Neurobiol
 , 
2002
, vol. 
68
 (pg. 
167
-
207
)
Bao
J
Li
JJ
Perl
ER
Differences in Ca2+ channels governing generation of miniature and evoked excitatory synaptic currents in spinal laminae I and II
J Neurosci
 , 
1998
, vol. 
18
 (pg. 
8740
-
8750
)
Barish
ME
Increases in intracellular calcium ion concentration during depolarization of cultured embryonic Xenopus spinal neurones
J Physiol
 , 
1991
, vol. 
444
 (pg. 
545
-
565
)
Beaulieu
JM
Gainetdinov
RR
The physiology, signaling, and pharmacology of dopamine receptors
Pharmacol Rev
 , 
2011
, vol. 
63
 (pg. 
182
-
217
)
Behr
J
Gloveli
T
Schmitz
D
Heinemann
U
Dopamine depresses excitatory synaptic transmission onto rat subicular neurons via presynaptic D1-like dopamine receptors
J Neurophysiol
 , 
2000
, vol. 
84
 (pg. 
112
-
119
)
Bessaih
T
Leresche
N
Lambert
RC
T current potentiation increases the occurrence and temporal fidelity of synaptically evoked burst firing in sensory thalamic neurons
Proc Natl Acad Sci USA
 , 
2008
, vol. 
105
 (pg. 
11376
-
11381
)
Booze
RM
Crisostomo
EA
Davis
JN
Beta-adrenergic receptors in the hippocampal and retrohippocampal regions of rats and guinea pigs: autoradiographic and immunohistochemical studies
Synapse
 , 
1993
, vol. 
13
 (pg. 
206
-
214
)
Boyajian
CL
Loughlin
SE
Leslie
FM
Anatomical evidence for alpha-2 adrenoceptor heterogeneity: differential autoradiographic distributions of [3H]rauwolscine and [3H]idazoxan in rat brain
J Pharmacol Exp Ther
 , 
1987
, vol. 
241
 (pg. 
1079
-
1091
)
Boyer
C
Lehouelleur
J
Sans
A
Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage-sensitive calcium channels
Eur J Neurosci
 , 
1998
, vol. 
10
 (pg. 
971
-
975
)
Carbone
E
Marcantoni
A
Giancippoli
A
Guido
D
Carabelli
V
T-type channels-secretion coupling: evidence for a fast low-threshold exocytosis
Pflugers Arch
 , 
2006
, vol. 
453
 (pg. 
373
-
383
)
Caruana
DA
Chapman
CA
Dopaminergic suppression of synaptic transmission in the lateral entorhinal cortex
Neural Plast
 , 
2008
, vol. 
2008
 pg. 
203514
 
Caruana
DA
Reed
SJ
Sliz
DJ
Chapman
CA
Inhibiting dopamine reuptake blocks the induction of long-term potentiation and depression in the lateral entorhinal cortex of awake rats
Neurosci Lett
 , 
2007
, vol. 
426
 (pg. 
6
-
11
)
Caruana
DA
Sorge
RE
Stewart
J
Chapman
CA
Dopamine has bidirectional effects on synaptic responses to cortical inputs in layer II of the lateral entorhinal cortex
J Neurophysiol
 , 
2006
, vol. 
96
 (pg. 
3006
-
3015
)
Chee
MJ
Price
CJ
Statnick
MA
Colmers
WF
Nociceptin/orphanin FQ suppresses the excitability of neurons in the ventromedial nucleus of the hypothalamus
J Physiol
 , 
2011
, vol. 
589
 (pg. 
3103
-
3114
)
Cornil
CA
Balthazart
J
Motte
P
Massotte
L
Seutin
V
Dopamine activates noradrenergic receptors in the preoptic area
J Neurosci
 , 
2002
, vol. 
22
 (pg. 
9320
-
9330
)
Cui
RJ
Roberts
BL
Zhao
H
Zhu
M
Appleyard
SM
Serotonin activates catecholamine neurons in the solitary tract nucleus by increasing spontaneous glutamate inputs
J Neurosci
 , 
2012
, vol. 
32
 (pg. 
16530
-
16538
)
Cutsuridis
V
Hasselmo
M
GABAergic contributions to gating, timing, and phase precession of hippocampal neuronal activity during theta oscillations
Hippocampus
 , 
2012
, vol. 
22
 (pg. 
1597
-
1621
)
Deng
PY
Lei
S
Bidirectional modulation of GABAergic transmission by cholecystokinin in hippocampal dentate gyrus granule cells of juvenile rats
J Physiol
 , 
2006
, vol. 
572
 (pg. 
425
-
442
)
Deng
PY
Lei
S
Long-term depression in identified stellate neurons of juvenile rat entorhinal cortex
J Neurophysiol
 , 
2007
, vol. 
97
 (pg. 
727
-
737
)
Deng
PY
Lei
S
Serotonin increases GABA release in rat entorhinal cortex by inhibiting interneuron TASK-3 K+ channels
Mol Cell Neurosci
 , 
2008
, vol. 
39
 (pg. 
273
-
284
)
Deng
PY
Porter
JE
Shin
HS
Lei
S
Thyrotropin-releasing hormone increases GABA release in rat hippocampus
J Physiol
 , 
2006
, vol. 
577
 (pg. 
497
-
511
)
Deng
PY
Xiao
Z
Jha
A
Ramonet
D
Matsui
T
Leitges
M
Shin
HS
Porter
JE
Geiger
JD
Lei
S
Cholecystokinin facilitates glutamate release by increasing the number of readily releasable vesicles and releasing probability
J Neurosci
 , 
2010
, vol. 
30
 (pg. 
5136
-
5148
)
Deng
PY
Xiao
Z
Lei
S
Distinct modes of modulation of GABAergic transmission by Group I metabotropic glutamate receptors in rat entorhinal cortex
Hippocampus
 , 
2010
, vol. 
20
 (pg. 
980
-
993
)
Dolcos
F
LaBar
KS
Cabeza
R
Remembering one year later: role of the amygdala and the medial temporal lobe memory system in retrieving emotional memories
Proc Natl Acad Sci USA
 , 
2005
, vol. 
102
 (pg. 
2626
-
2631
)
Dong
Y
Cooper
D
Nasif
F
Hu
XT
White
FJ
Dopamine modulates inwardly rectifying potassium currents in medial prefrontal cortex pyramidal neurons
J Neurosci
 , 
2004
, vol. 
24
 (pg. 
3077
-
3085
)
Erickson
SL
Akil
M
Levey
AI
Lewis
DA
Postnatal development of tyrosine hydroxylase- and dopamine transporter-immunoreac-tive axons in monkey rostral entorhinal cortex
Cereb Cortex
 , 
1998
, vol. 
8
 (pg. 
415
-
427
)
Falkai
P
Bogerts
B
Rozumek
M
Limbic pathology in schizophrenia: the entorhinal region—a morphometric study
Biol Psychiatry
 , 
1988
, vol. 
24
 (pg. 
515
-
521
)
Fallon
JH
Koziell
DA
Moore
RY
Catecholamine innervation of the basal forebrain. II. Amygdala, suprarhinal cortex and entorhinal cortex
J Comp Neurol
 , 
1978
, vol. 
180
 (pg. 
509
-
532
)
Ford
CP
Phillips
PE
Williams
JT
The time course of dopamine transmission in the ventral tegmental area
J Neurosci
 , 
2009
, vol. 
29
 (pg. 
13344
-
13352
)
Gioanni
Y
Thierry
AM
Glowinski
J
Tassin
JP
Alpha1-adrenergic, D1, and D2 receptors interactions in the prefrontal cortex: implications for the modality of action of different types of neuroleptics
Synapse
 , 
1998
, vol. 
30
 (pg. 
362
-
370
)
Gorelova
N
Seamans
JK
Yang
CR
Mechanisms of dopamine activation of fast-spiking interneurons that exert inhibition in rat prefrontal cortex
J Neurophysiol
 , 
2002
, vol. 
88
 (pg. 
3150
-
3166
)
Govindaiah
G
Wang
Y
Cox
CL
Dopamine enhances the excitability of somatosensory thalamocortical neurons
Neuroscience
 , 
2010
, vol. 
170
 (pg. 
981
-
991
)
Haist
F
Bowden Gore
J
Mao
H
Consolidation of human memory over decades revealed by functional magnetic resonance imaging
Nat Neurosci
 , 
2001
, vol. 
4
 (pg. 
1139
-
1145
)
Hamilton
TJ
Wheatley
BM
Sinclair
DB
Bachmann
M
Larkum
ME
Colmers
WF
Dopamine modulates synaptic plasticity in dendrites of rat and human dentate granule cells
Proc Natl Acad Sci USA
 , 
2010
, vol. 
107
 (pg. 
18185
-
18190
)
Hemby
SE
Trojanowski
JQ
Ginsberg
SD
Neuron-specific age-related decreases in dopamine receptor subtype mRNAs
J Comp Neurol
 , 
2003
, vol. 
456
 (pg. 
176
-
183
)
Hidaka
H
Fusaric (5-butylpicolinic) acid, an inhibitor of dopamine beta-hydroxylase, affects serotonin and noradrenaline
Nature
 , 
1971
, vol. 
231
 (pg. 
54
-
55
)
Huang
Q
Zhou
D
Chase
K
Gusella
JF
Aronin
N
DiFiglia
M
Immunohistochemical localization of the D1 dopamine receptor in rat brain reveals its axonal transport, pre- and postsynaptic localization, and prevalence in the basal ganglia, limbic system, and thalamic reticular nucleus
Proc Natl Acad Sci USA
 , 
1992
, vol. 
89
 (pg. 
11988
-
11992
)
Hyman
BT
Van Hoesen
GW
Damasio
AR
Barnes
CL
Alzheimer's disease: cell-specific pathology isolates the hippocampal formation
Science
 , 
1984
, vol. 
225
 (pg. 
1168
-
1170
)
Jensen
LJ
Salomonsson
M
Jensen
BL
Holstein-Rathlou
NH
Depolarization-induced calcium influx in rat mesenteric small arterioles is mediated exclusively via mibefradil-sensitive calcium channels
Br J Pharmacol
 , 
2004
, vol. 
142
 (pg. 
709
-
718
)
Joyal
CC
Laakso
MP
Tiihonen
J
Syvalahti
E
Vilkman
H
Laakso
A
Alakare
B
Rakkolainen
V
Salokangas
RK
Hietala
J
A volumetric MRI study of the entorhinal cortex in first episode neuroleptic-naive schizophrenia
Biol Psychiatry
 , 
2002
, vol. 
51
 (pg. 
1005
-
1007
)
Kassack
MU
Höfgen
B
Decker
M
Eckstein
N
Lehmann
J
Pharmacological characterization of the benz[d]indolo[2,3-g]azecine LE300, a novel type of a nanomolar dopamine receptor antagonist
Naunyn Schmiedebergs Arch Pharmacol
 , 
2002
, vol. 
366
 (pg. 
543
-
550
)
Kawai
F
Miyachi
E
Enhancement by T-type Ca2+ currents of odor sensitivity in olfactory receptor cells
J Neurosci
 , 
2001
, vol. 
21
 pg. 
RC144
 
Kotzbauer
PT
Trojanowsk
JQ
Lee
VM
Lewy body pathology in Alzheimer's disease
J Mol Neurosci
 , 
2001
, vol. 
17
 (pg. 
225
-
232
)
Kurian
MA
Gissen
P
Smith
M
Heales
S
Jr
Clayton
PT
The monoamine neurotransmitter disorders: an expanding range of neurological syndromes
Lancet Neurol
 , 
2011
, vol. 
10
 (pg. 
721
-
733
)
Kuzhikandathil
EV
Oxford
GS
Classic D1 dopamine receptor antagonist R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) directly inhibits G protein-coupled inwardly rectifying potassium channels
Mol Pharmacol
 , 
2002
, vol. 
62
 (pg. 
119
-
126
)
Lazou
A
Markou
T
Zioga
M
Vasara
E
Efstathiou
A
Gaitanaki
C
Dopamine mimics the cardioprotective effect of ischemic preconditioning via activation of alpha1-adrenoceptors in the isolated rat heart
Physiol Res
 , 
2006
, vol. 
55
 (pg. 
1
-
8
)
Lee
TL
Hsu
CT
Yen
ST
Lai
CW
Cheng
JT
Activation of beta3-adrenoceptors by exogenous dopamine to lower glucose uptake into rat adipocytes
J Auton Nerv Syst
 , 
1998
, vol. 
74
 (pg. 
86
-
90
)
Leedham
JA
Pennefather
JN
Selectivities of some agonists acting at alpha 1- and alpha 2-adrenoreceptors in the rat vas deferens
J Auton Pharmacol
 , 
1986
, vol. 
6
 (pg. 
39
-
46
)
Lei
S
Deng
PY
Porter
JE
Shin
HS
Adrenergic facilitation of GABAergic transmission in rat entorhinal cortex
J Neurophysiol
 , 
2007
, vol. 
98
 (pg. 
2868
-
2877
)
Leviel
V
Dopamine release mediated by the dopamine transporter, facts and consequences
J Neurochem
 , 
2011
, vol. 
118
 (pg. 
475
-
489
)
Lin
Y
Quartermain
D
Dunn
AJ
Weinshenker
D
Stone
EA
Possible dopaminergic stimulation of locus coeruleus alpha1-adrenoceptors involved in behavioral activation
Synapse
 , 
2008
, vol. 
62
 (pg. 
516
-
523
)
Loretan
K
Bissiere
S
Luthi
A
Dopaminergic modulation of spontaneous inhibitory network activity in the lateral amygdala
Neuropharmacology
 , 
2004
, vol. 
47
 (pg. 
631
-
639
)
Lu
J
Dalton
JFt
Stokes
DR
Calabrese
RL
Functional role of Ca2+ currents in graded and spike-mediated synaptic transmission between leech heart interneurons
J Neurophysiol
 , 
1997
, vol. 
77
 (pg. 
1779
-
1794
)
Mayne
EW
Craig
MT
McBain
CJ
Paulsen
O
Dopamine suppresses persistent network activity via D(1) -like dopamine receptors in rat medial entorhinal cortex
Eur J Neurosci
 , 
2013
, vol. 
37
 (pg. 
1242
-
1247
)
McConathy
J
Owens
MJ
Kilts
CD
Malveaux
EJ
Camp
VM
Votaw
JR
Nemeroff
CB
Goodman
MM
Synthesis and biological evaluation of [11C]talopram and [11C]talsupram: candidate PET ligands for the norepinephrine transporter
Nucl Med Biol
 , 
2004
, vol. 
31
 (pg. 
705
-
718
)
Munsch
T
Yanagawa
Y
Obata
K
Pape
HC
Dopaminergic control of local interneuron activity in the thalamus
Eur J Neurosci
 , 
2005
, vol. 
21
 (pg. 
290
-
294
)
Nagatsu
T
Hidaka
H
Kuzuya
H
Takeya
K
Umezawa
H
Inhibition of dopamine beta-hydroxylase by fusaric acid (5-butylpicolinic acid) in vitro and in vivo
Biochem Pharmacol
 , 
1970
, vol. 
19
 (pg. 
35
-
44
)
Ooi
H
Colucci
WS
Hardman
JG
Limbird
LE
Gilman
AG
Pharmacological treatment of heart failure
The pharmacological basis of therapeutics
 , 
2001
10th ed
New York
McGraw-Hill
pg. 
901
 
Ouedraogo
L
Magnon
M
Sawadogo
L
Tricoche
R
Receptors involved in the positive inotropic action induced by dopamine on the ventricle of a 7-day-old chick embryo heart
Fundam Clin Pharmacol
 , 
1998
, vol. 
12
 (pg. 
133
-
142
)
Palkovits
M
Zaborszky
L
Brownstein
MJ
Fekete
MI
Herman
JP
Kanyicska
B
Distribution of norepinephrine and dopamine in cerebral cortical areas of the rat
Brain Res Bull
 , 
1979
, vol. 
4
 (pg. 
593
-
601
)
Pan
ZH
Hu
HJ
Perring
P
Andrade
R
T-type Ca(2+) channels mediate neurotransmitter release in retinal bipolar cells
Neuron
 , 
2001
, vol. 
32
 (pg. 
89
-
98
)
Phillips
AG
Vacca
G
Ahn
S
A top-down perspective on dopamine, motivation and memory
Pharmacol Biochem Behav
 , 
2008
, vol. 
90
 (pg. 
236
-
249
)
Podda
MV
Riccardi
E
D'Ascenzo
M
Azzena
GB
Grassi
C
Dopamine D1-like receptor activation depolarizes medium spiny neurons of the mouse nucleus accumbens by inhibiting inwardly rectifying K+ currents through a cAMP-dependent protein kinase A-independent mechanism
Neuroscience
 , 
2010
, vol. 
167
 (pg. 
678
-
690
)
Pralong
E
Jones
RS
Interactions of dopamine with glutamate- and GABA-mediated synaptic transmission in the rat entorhinal cortex in vitro
Eur J Neurosci
 , 
1993
, vol. 
5
 (pg. 
760
-
767
)
Pralong
E
Magistretti
PJ
Noradrenaline increases K-conductance and reduces glutamatergic transmission in the mouse entorhinal cortex by activation of alpha 2-adrenoreceptors
Eur J Neurosci
 , 
1995
, vol. 
7
 (pg. 
2370
-
2378
)
Pralong
E
Magistretti
PJ
Noradrenaline reduces synaptic responses in normal and tottering mouse entorhinal cortex via alpha 2 receptors
Neurosci Lett
 , 
1994
, vol. 
179
 (pg. 
145
-
148
)
Prasad
KM
Patel
AR
Muddasani
S
Sweeney
J
Keshavan
MS
The entorhinal cortex in first-episode psychotic disorders: a structural magnetic resonance imaging study
Am J Psychiatry
 , 
2004
, vol. 
161
 (pg. 
1612
-
1619
)
Rajfer
SI
Borow
KM
Lang
RM
Neumann
A
Carroll
JD
Effects of dopamine on left ventricular afterload and contractile state in heart failure: relation to the activation of beta 1-adrenoceptors and dopamine receptors
J Am Coll Cardiol
 , 
1988
, vol. 
12
 (pg. 
498
-
506
)
Ramanathan
G
Cilz
NI
Kurada
L
Hu
B
Wang
X
Lei
S
Vasopressin facilitates GABAergic transmission in rat hippocampus via activation of V1A receptors
Neuropharmacology
 , 
2012
, vol. 
63
 (pg. 
1218
-
1226
)
Rey
E
Hernandez-Diaz
FJ
Abreu
P
Alonso
R
Tabares
L
Dopamine induces intracellular Ca2+ signals mediated by alpha1B-adrenoceptors in rat pineal cells
Eur J Pharmacol
 , 
2001
, vol. 
430
 (pg. 
9
-
17
)
Richfield
EK
Young
AB
Penney
JB
Comparative distributions of dopamine D-1 and D-2 receptors in the cerebral cortex of rats, cats, and monkeys
J Comp Neurol
 , 
1989
, vol. 
286
 (pg. 
409
-
426
)
Rivera
A
Penafiel
A
Megias
M
Agnati
LF
Lopez-Tellez
JF
Gago
B
Gutierrez
A
de la Calle
A
Fuxe
K
Cellular localization and distribution of dopamine D(4) receptors in the rat cerebral cortex and their relationship with the cortical dopaminergic and noradrenergic nerve terminal networks
Neuroscience
 , 
2008
, vol. 
155
 (pg. 
997
-
1010
)
Robbins
J
Wakakuwa
K
Ikeda
H
Noradrenaline action on cat retinal ganglion cells is mediated by dopamine (D2) receptors
Brain Res
 , 
1988
, vol. 
438
 (pg. 
52
-
60
)
Rosenkranz
JA
Johnston
D
Dopaminergic regulation of neuronal excitability through modulation of Ih in layer V entorhinal cortex
J Neurosci
 , 
2006
, vol. 
26
 (pg. 
3229
-
3244
)
Rosenkranz
JA
Johnston
D
State-dependent modulation of amygdala inputs by dopamine-induced enhancement of sodium currents in layer V entorhinal cortex
J Neurosci
 , 
2007
, vol. 
27
 (pg. 
7054
-
7069
)
Rothman
RB
Baumann
MH
Dersch
CM
Romero
DV
Rice
KC
Carroll
FI
Partilla
JS
Amphetamine-type central nervous system stimulants release norepinephrine more potently than they release dopamine and serotonin
Synapse
 , 
2001
, vol. 
39
 (pg. 
32
-
41
)
Sara
SJ
The locus coeruleus and noradrenergic modulation of cognition
Nat Rev Neurosci
 , 
2009
, vol. 
10
 (pg. 
211
-
223
)
Savasta
M
Dubois
A
Scatton
B
Autoradiographic localization of D1 dopamine receptors in the rat brain with [3H]SCH 23390
Brain Res
 , 
1986
, vol. 
375
 (pg. 
291
-
301
)
Seamans
JK
Gorelova
N
Durstewitz
D
Yang
CR
Bidirectional dopamine modulation of GABAergic inhibition in prefrontal cortical pyramidal neurons
J Neurosci
 , 
2001
, vol. 
21
 (pg. 
3628
-
3638
)
Shankar
H
Murugappan
S
Kim
S
Jin
J
Ding
Z
Wickman
K
Kunapuli
SP
Role of G protein-gated inwardly rectifying potassium channels in P2Y12 receptor-mediated platelet functional responses
Blood
 , 
2004
, vol. 
104
 (pg. 
1335
-
1343
)
Smith
CC
Greene
RW
CNS dopamine transmission mediated by noradrenergic innervation
J Neurosci
 , 
2012
, vol. 
32
 (pg. 
6072
-
6080
)
Sosulina
L
Schwesig
G
Seifert
G
Pape
HC
Neuropeptide Y activates a G-protein-coupled inwardly rectifying potassium current and dampens excitability in the lateral amygdala
Mol Cell Neurosci
 , 
2008
, vol. 
39
 (pg. 
491
-
498
)
Spencer
SS
Spencer
DD
Entorhinal-hippocampal interactions in medial temporal lobe epilepsy
Epilepsia
 , 
1994
, vol. 
35
 (pg. 
721
-
727
)
Squire
LR
Stark
CE
Clark
RE
The medial temporal lobe
Annu Rev Neurosci
 , 
2004
, vol. 
27
 (pg. 
279
-
306
)
Steffenach
HA
Witter
M
Moser
MB
Moser
EI
Spatial memory in the rat requires the dorsolateral band of the entorhinal cortex
Neuron
 , 
2005
, vol. 
45
 (pg. 
301
-
313
)
Stenkamp
K
Heinemann
U
Schmitz
D
Dopamine suppresses stimulus-induced field potentials in layer III of rat medial entorhinal cortex
Neurosci Lett
 , 
1998
, vol. 
255
 (pg. 
119
-
121
)
Steward
O
Scoville
SA
Cells of origin of entorhinal cortical afferents to the hippocampus and fascia dentata of the rat
J Comp Neurol
 , 
1976
, vol. 
169
 (pg. 
347
-
370
)
Stuchlik
A
Petrasek
T
Vales
K
Dopamine D2 receptors and alpha1-adrenoceptors synergistically modulate locomotion and behavior of rats in a place avoidance task
Behav Brain Res
 , 
2008
, vol. 
189
 (pg. 
139
-
144
)
Tarazi
FI
Tomasini
EC
Baldessarini
RJ
Postnatal development of dopamine D1-like receptors in rat cortical and striatolimbic brain regions: an autoradiographic study
Dev Neurosci
 , 
1999
, vol. 
21
 (pg. 
43
-
49
)
Unnerstall
JR
Fernandez
I
Orensanz
LM
The alpha-adrenergic receptor: radiohistochemical analysis of functional characteristics and biochemical differences
Pharmacol Biochem Behav
 , 
1985
, vol. 
22
 (pg. 
859
-
874
)
Unnerstall
JR
Kopajtic
TA
Kuhar
MJ
Distribution of alpha 2 agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents
Brain Res
 , 
1984
, vol. 
319
 (pg. 
69
-
101
)
Varnai
P
Osipenko
ON
Vizi
ES
Spat
A
Activation of calcium current in voltage-clamped rat glomerulosa cells by potassium ions
J Physiol
 , 
1995
, vol. 
483
 
Pt 1
(pg. 
67
-
78
)
Wadenberg
ML
Hertel
P
Fernholm
R
Hygge Blakeman
K
Ahlenius
S
Svensson
TH
Enhancement of antipsychotic-like effects by combined treatment with the alpha1-adrenoceptor antagonist prazosin and the dopamine D2 receptor antagonist raclopride in rats
J Neural Transm
 , 
2000
, vol. 
107
 (pg. 
1229
-
1238
)
Wang
S
Chen
X
Kurada
L
Huang
Z
Lei
S
Activation of group II metabotropic glutamate receptors inhibits glutamatergic transmission in the rat entorhinal cortex via reduction of glutamate release probability
Cereb Cortex
 , 
2012
, vol. 
22
 (pg. 
584
-
594
)
Wang
S
Zhang
AP
Kurada
L
Matsui
T
Lei
S
Cholecystokinin facilitates neuronal excitability in the entorhinal cortex via activation of TRPC-like channels
J Neurophysiol
 , 
2011
, vol. 
106
 (pg. 
1515
-
1524
)
Weiner
DM
Levey
AI
Sunahara
RK
Niznik
HB
O'Dowd
BF
Seeman
P
Brann
MR
D1 and D2 dopamine receptor mRNA in rat brain
Proc Natl Acad Sci USA
 , 
1991
, vol. 
88
 (pg. 
1859
-
1863
)
Wilcox
BJ
Unnerstall
JR
Identification of a subpopulation of neuropeptide Y-containing locus coeruleus neurons that project to the entorhinal cortex
Synapse
 , 
1990
, vol. 
6
 (pg. 
284
-
291
)
Witkowski
G
Szulczyk
B
Rola
R
Szulczyk
P
D(1) dopaminergic control of G protein-dependent inward rectifier K(+) (GIRK)-like channel current in pyramidal neurons of the medial prefrontal cortex
Neuroscience
 , 
2008
, vol. 
155
 (pg. 
53
-
63
)
Witter
MP
Groenewegen
HJ
Lopes da Silva
FH
Lohman
AH
Functional organization of the extrinsic and intrinsic circuitry of the parahippocampal region
Prog Neurobiol
 , 
1989
, vol. 
33
 (pg. 
161
-
253
)
Witter
MP
Naber
PA
van Haeften
T
Machielsen
WC
Rombouts
SA
Barkhof
F
Scheltens
P
Lopes da Silva
FH
Cortico-hippocampal communication by way of parallel parahippocampal-subicular pathways
Hippocampus
 , 
2000
, vol. 
10
 (pg. 
398
-
410
)
Witter
MP
Wouterlood
FG
Naber
PA
Van Haeften
T
Anatomical organization of the parahippocampal-hippocampal network
Ann N Y Acad Sci
 , 
2000
, vol. 
911
 (pg. 
1
-
24
)
Xiao
Z
Deng
PY
Rojanathammanee
L
Yang
C
Grisanti
L
Permpoonputtana
K
Weinshenker
D
Doze
VA
Porter
JE
Lei
S
Noradrenergic depression of neuronal excitability in the entorhinal cortex via activation of TREK-2 K+ channels
J Biol Chem
 , 
2009
, vol. 
284
 (pg. 
10980
-
10991
)
Xiao
Z
Deng
PY
Yang
C
Lei
S
Modulation of GABAergic transmission by muscarinic receptors in the entorhinal cortex of juvenile rats
J Neurophysiol
 , 
2009
, vol. 
102
 (pg. 
659
-
669
)
Zhang
WP
Ouyang
M
Thomas
SA
Potency of catecholamines and other L-tyrosine derivatives at the cloned mouse adrenergic receptors
Neuropharmacology
 , 
2004
, vol. 
47
 (pg. 
438
-
449
)
Zhou
FM
Hablitz
JJ
Dopamine modulation of membrane and synaptic properties of interneurons in rat cerebral cortex
J Neurophysiol
 , 
1999
, vol. 
81
 (pg. 
967
-
976
)