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Adrenergic Facilitation of GABAergic Transmission in Rat Entorhinal Cortex

¹Department of Pharmacology, Physiology and Therapeutics, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota; and ²Center for Neural Science, Korea Institute of Science and Technology, Seoul, Republic of Korea

Submitted 19 June 2007; accepted in final form 5 September 2007

	ABSTRACT

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Whereas the entorhinal cortex (EC) receives noradrenergic innervations from the locus coeruleus of the pons and expresses adrenergic receptors, the function of norepinephrine (NE) in the EC is still elusive. We examined the effects of NE on GABA_A receptor–mediated synaptic transmission in the superficial layers of the EC. Application of NE dose-dependently increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from the principal neurons in layer II/III through activation of ₁ adrenergic receptors. NE increased the frequency and not the amplitude of miniature IPSCs (mIPSCs) recorded in the presence of TTX, suggesting that NE increases presynaptic GABA release with no effects on postsynaptic GABA_A receptors. Application of Ca²⁺ channel blockers (Cd²⁺ and Ni²⁺), omission of Ca²⁺ in the extracellular solution, or replacement of extracellular Na⁺ with N-methyl-D-glucamine (NMDG) failed to alter NE-induced increase in mIPSC frequency, suggesting that Ca²⁺ influx through voltage-gated Ca²⁺ or other cationic channels is not required. Application of BAPTA-AM, thapsigargin, and ryanodine did not change NE-induced increase in mIPSC frequency, suggesting that Ca²⁺ release from intracellular stores is not necessary for NE-induced increase in GABA release. Whereas ₁ receptors are coupled to G_q/11 resulting in activation of the phospholipase C (PLC) pathway, NE-mediated facilitation of GABAergic transmission was independent of PLC, protein kinase C, and tyrosine kinase activities. Our results suggest that NE-mediated facilitation of GABAergic function contributes to its antiepileptic effects in the EC.

	INTRODUCTION

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The entorhinal cortex (EC) mediates the majority of connections between the hippocampus and other cortical areas (Witter et al. 1989, 2000). Sensory inputs converge onto the superficial layers (layers I–III) of the EC (Burwell 2000), which give rise to dense projections to the hippocampus; the axons of the stellate neurons in layer II of the EC form the perforant path that innervates the dentate gyrus and CA3 (Steward and Scoville 1976), whereas pyramidal neurons in layer II/III provide the primary input to CA1 regions (Steward and Scoville 1976; Witter et al. 2000). Moreover, neurons in the deep layers of the EC (layers IV–VI) relay a large portion of hippocampal output projections back to the superficial layers of the EC (Dolorfo and Amaral 1998a,b; Kohler 1986; van Haeften et al. 2003) and to other cortical areas (Witter et al. 1989). The EC is part of a network that is closely related to the consolidation and recall of memories (for reviews, see Dolcos et al. 2005; Haist et al. 2001; Squire et al. 2004; Steffenach et al. 2005), Alzheimer's disease (Hyman et al. 1984; Kotzbauer et al. 2001), schizophrenia (Arnold et al. 1991; Falkai et al. 1988; Joyal et al. 2002; Prasad et al. 2004), and temporal lobe epilepsy (Spencer and Spencer 1994).

The EC receives innervations from the cortical mantle and from the brain stem. The locus coeruleus sends strong noradrenergic projections to the EC (Fallon et al. 1978; Palkovits et al. 1979; Wilcox and Unnerstall 1990). The EC also expresses ₁ (Stanton et al. 1987), ₂ (Boyajian et al. 1987; Unnerstall et al. 1984, 1985), and (Booze et al. 1993) adrenergic receptors. In accordance with the structural innervations of noradrenergic fibers and the expression of adrenergic receptors in the EC, application of norepinephrine (NE) inhibits excitatory synaptic transmission in the EC (Pralong and Magistretti 1994, 1995) and reduces epileptiform discharges induced by bicuculline (Stoop et al. 2000) through ₂ receptors. Furthermore, NE has been reported to block low Mg²⁺-induced epileptiform activity through ₁ receptors in the EC (Stanton et al. 1987). Because bicuculline-induced epileptic model is produced by inhibition of GABAergic transmission, whereas low Mg²⁺-induced epileptic model is caused by an overactivation of N-methyl-D-aspartate (NMDA) type of glutamate receptors, these results suggest that NE modulates inhibitory and excitatory synaptic transmission through distinct adrenergic receptors. However, the effects of NE on inhibitory synaptic transmission have never been determined. In this study, we examined the effects of NE on GABAergic transmission in the EC. Our results indicate that NE increases GABAergic transmission in the superficial layers of the EC through activation of ₁ receptors. NE-mediated increase in GABA release is independent of Ca²⁺, phospholipase C (PLC), protein kinase C (PKC), and tyrosine kinase activities. NE-mediated facilitation of GABAergic function likely contributes to its antiepileptic effects in the EC.

	METHODS

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Slice preparation

Horizontal brain slices (400 µm) including the EC, subiculum, and hippocampus were cut using a vibrating blade microtome (VT1000S, Leica, Wetzlar, Germany), usually from 13- to 20-day-old Sprague-Dawley rats as described previously (Deng and Lei 2006, 2007; Deng et al. 2006, 2007). After being deeply anesthetized with isoflurane, rats were decapitated, and their brains were dissected out in ice-cold saline solution that contained (in mM) 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 5.0 MgCl₂, and 10 glucose, saturated with 95% O₂-5% CO₂, pH 7.4. Slices were initially incubated in the preceding solution at 35°C for 40 min for recovery and kept at room temperature (24°C) until use. All animal procedures conformed to the guidelines approved by the University of North Dakota Animal Care and Use Committee.

Recordings of spontaneous, miniature, and evoked GABA_A receptor–mediated IPSCs

Whole cell patch-clamp recordings using two Multiclamp 700B amplifiers (Molecular Devices, Sunnyvale, CA) in voltage-clamp mode were made from the principal neurons in layer II/III of the EC visually identified with infrared video microscopy (BX51WI, Olympus, Tokyo, Japan) and differential interference contrast optics (Deng and Lei 2007; Deng et al. 2007). The recording electrodes were filled with the following solution (in mM): 100 caesium gluconate, 0.6 EGTA, 5 MgCl₂, 8 NaCl, 2 ATP₂Na, 0.3 GTPNa, 40 HEPES, and 1 QX-314; pH 7.3. The extracellular solution contained (in mM) 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 1.5 MgCl₂, 2.5 CaCl₂, and 10 glucose, saturated with 95% O₂-5% CO₂; pH 7.4. To record GABA_A receptor–mediated spontaneous inhibitory postsynaptic currents (sIPSCs), the external solution was supplemented with DL-2-amino-5-phosphonovaleric acid (D-APV; 100 µM) and 6,7-dinitroquinoxaline-2,3(1H, 4H)-dione (DNQX; 10 µM) to block NMDA and AMPA receptor–mediated responses, respectively. Under these conditions, the recorded inhibitory currents had a reversal potential of approximately –30 mV and were completely blocked by bicuculline methobromide (10 µM), confirming that they were mediated by GABA_A receptors. Usually sIPSCs were recorded at a holding potential of +30 mV (Deng and Lei 2006; Deng et al. 2006; Lei and McBain 2003). Miniature IPSCs (mIPSCs) were recorded by including TTX (1 µM) in the preceding external solution to block action potential-dependent responses. Evoked IPSCs were recorded from stellate and pyramidal neurons in the EC using the same internal and external solution at a holding potential of +30 mV by placing a stimulation electrode locally. Synaptic responses were evoked at 0.2 Hz by low-intensity stimulation (80- to 100-µs duration; 10- to 40-µA intensity) using a constant-current isolation unit (A360, World Precision Instrument, Sarasota, FL) connected to a patch electrode filled with oxygenated extracellular solution. Series resistance was rigorously monitored by the delivery of 5-mV voltage steps after each evoked current. Experiments were discontinued if the series resistance changed by >10%. Data were filtered at 2 kHz, digitized at 10 kHz, and acquired on-line using pCLAMP 9 (Clampex) software (Molecular Devices). The recorded sIPSCs and mIPSCs were subsequently analyzed by Mini Analysis 6.0.1 (Synaptosoft, Decatur, GA). Each detected event was inspected visually to exclude obvious artifacts before analysis. The threshold for detection was set to 3 times the SD of the noise as recorded in an event-free stretch of data (Clements and Bekkers 1997). Mean amplitude, frequency, cumulative amplitude, and frequency histograms were calculated by this program. NE and other drugs were bath applied. To avoid potential desensitization induced by repeated applications of NE, one slice was limited to only one application of NE. 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.

Recordings of action potentials or firing activity in whole cell or cell-attached patches

Whole cell recordings in current clamp were used to record action potentials from the principal neurons in layer II/III and interneurons in layer III of the EC. The intracellular solution contained (in mM) 130 K⁺-gluconate, 0.5 EGTA, 2 MgCl₂, 5 NaCl, 2 ATP₂Na, 0.4 GTPNa, and 10 HEPES; pH 7.4. Biocytin (0.2%) was added in the intracellular solution for the recordings of action potentials from interneurons in layer III for ad hoc histological identification. Because dialysis of K⁺-containing internal solution into cells can change the resting membrane potential and influence action potential firing, we waited for 15 min after the formation of whole cell recordings to allow the resting membrane potential to stabilize. Usually, for most of the cells, a positive current injection was needed to bring the membrane potential to approximately –50 mV to induce action potential firing. Cell-attached patches were used in some experiments to record the firing activity of interneurons in layer III. The pipettes were filled with the above K⁺-gluconate solution. NE was applied after the action potentials or the firing activity had been stable for 510 min. The frequency of the action potentials or firing activity was calculated by Mini Analysis 6.0.1.

Recordings of holding current

Holding current at –55 mV was recorded from interneurons in layer III in the extracellular solution containing TTX (1 µM) to block action potential firing. The intracellular solution was the above K⁺-gluconate solution containing 0.2% biocytin. Because gradual dialysis of K⁺ into cells changed the holding current, we began our recordings after waiting for 15 min from the formation of whole cell configuration. Holding currents at –55 mV were recorded every 3 s and averaged per minute. We subtracted the average of the holding current recorded for the last minute before the application of NE from those recorded at different time-points to zero the basal level of the holding current for better comparison.

Histological staining of interneurons

After recordings, slices were fixed in 0.1 M PBS containing 4% paraformaldehyde and 0.2% picric acid for 24 h at 4°C. After an extensive wash in 0.1 M PBS, slices were incubated with Texas red–conjugated streptavidin (1:200) for 2 h at room temperature. After wash, slices were mounted on slides and coverslipped. Slides were visualized with an Olympus Fluoview 300 confocal microscope and photographed.

Breeding and genotyping of mutant mice

Heterozygous mating pairs (F1 hybrid crosses from 129 PLC-1^+/– x C57BL/6J PLC-1^+/–) were obtained from the Korea Institute of Science and Technology. The breeders were used to derive wild-type, heterozygous, and homozygous pups for experimental analysis. PCR genotyping from purified genomic DNA was performed as described previously (Deng et al. 2006; Kim et al. 1997).

Data analysis

Data are presented as the means ± SE. Concentration–response curve of NE was fit by Hill equation: I = I_max x (1/{1 + [EC₅₀/(ligand)]ⁿ]), where I_max is the maximum response, EC₅₀ is the concentration of ligand producing a half-maximal response, and n is the Hill coefficient. Student's paired or unpaired t-test or ANOVA was used for statistical analysis as appropriate; P values are reported throughout the text, and significance was set as P < 0.05. For sIPSC or mIPSC cumulative probability plots, events recorded 5 min before and 5 min after reaching the maximal effect of NE were selected. Same bin size (25 ms for frequency and 2 pA for amplitude) was used to analyze data from control and NE treatment. Kolmogorov-Smirnoff test was used to assess the significance of the cumulative probability plots. N in the text represents the cells examined.

Chemicals

Corynanthine, yohimbine, propranolol, genistein, and U73122 [GenBank] were purchased from TOCRIS (Ellisville, MO). Calphostin C and Ro318220 were from BIOMOL (Plymouth Meeting, PA). 1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (edelfosine) was purchased from Calbiochem (Darmstadt, Germany). NE and other chemicals were products of Sigma-Aldrich (St. Louis, MO).

	RESULTS

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NE increases the frequency and amplitude of sIPSCs

Stellate and pyramidal neurons are the two major types of neurons in the superficial layers of the EC. In this study, we identified these two types of neurons by their morphology and location because the characteristic electrophysiological property of stellate neurons (depolarizing voltage sag in response to hyperpolarizing current pulses; Deng and Lei 2007; Deng et al. 2007) could not be observed when Cs⁺ and QX-314 were included in the intracellular solution to record GABA_A receptor–mediated synaptic currents. Stellate neurons are usually located in layer II or the border of layer II and III, and they have larger and polygonal soma with variable number of main dendrites radiating out from the cell body, but are devoid of a clearly dominant dendrite. Pyramidal neurons have a pyramidal or elongated soma with dendrites orientated in a bidirectional way; one (sometimes 2) thick apical dendrite that runs to the surface of the cortex and several (3–5) basal dendrites extending toward the deeper layers. We recorded sIPSCs from both stellate and pyramidal neurons in layer II/III of the EC. Application of NE (100 µM) significantly increased the frequency (157 ± 11% of control, P < 0.001; Fig. 1, A–C) and amplitude (152 ± 12% of control, P = 0.011; Fig. 1D) of sIPSCs in five of five stellate neurons examined. Similarly, application of NE (100 µM) significantly increased the frequency (163 ± 12% of control, P < 0.001; Fig. 1, E–G) and amplitude (162 ± 19% of control, P = 0.03; Fig. 1H) of sIPSCs in five of five pyramidal neurons examined. Because there were indistinguishable differences for NE-induced increases in sIPSC frequency (P = 0.69, Student's unpaired t-test) and amplitude (P = 0.66, Student's unpaired t-test) recorded from stellate neurons and pyramidal neurons, we performed the rest of the experiments on both stellate and pyramidal neurons. The frequency of sIPSCs after application of NE became so high (Fig. 1, A and E) that it prevented reliable comparison of the decay kinetics of sIPSCs. The EC₅₀ value was measured to be 4.4 and 5.0 µM when the percentage of increase in frequency (Fig. 2A) or amplitude (Fig. 2B) was plotted versus the concentrations of NE, respectively.

View larger version (31K): [in this window] [in a new window]   FIG. 1. Norepinephrine (NE) increases the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from stellate neurons and pyramidal neurons in the entorhinal cortex (EC). A: sIPSCs recorded before (top) and during (bottom) application of NE (100 µM). Events in middle of 3rd trace were enlarged to show individual sIPSCs. B: pooled time-course of sIPSC frequency from 5 stellate neurons. C: cumulative probability of sIPSC frequency before and during application of NE from 5 stellate neurons. Note that NE increased sIPSC frequency (reduction in intervals of events). D: cumulative probability of sIPSC amplitude before and during application of NE from 5 stellate neurons. E–H: data from 5 pyramidal neurons were arranged in the same way.

View larger version (40K): [in this window] [in a new window]   FIG. 2. NE dose-dependently increases sIPSC frequency and amplitude through activation of 1 receptors. A: concentration–response curve by plotting percentage of increase in frequency vs. concentrations of NE. Numbers in parentheses are number of cells recoded. B: concentration–response curve by plotting percentage of increase in amplitude vs. concentrations of NE. Numbers in parentheses are number of cells recoded. C: application of corynanthine (100 µM), a 1 receptor blocker, blocked NE-induced increase in sIPSC frequency (n = 5). D: application of yohimbine (100 µM), a 2 receptor blocker, failed to change NE-induced increase in sIPSC frequency (n = 5). E: application of propranolol (100 µM), a receptor blocker, failed to change NE-induced increase in sIPSC frequency (n = 5). F: application of phenylephrine (100 µM), a 1 receptor agonist, increased sIPSC frequency (n = 5).

Involvement of ₁ receptors

NE possesses high potency for ₁ and ₂ but has weak activity on ₁ adrenergic receptors. We next examined the roles of these receptors in the effects of NE on sIPSCs. Application of a specific ₁ receptor blocker, corynathine (100 µM), completely blocked the NE-induced increase in sIPSC frequency (101 ± 7% of control, n = 5, P = 0.91; Fig. 2C) and amplitude (97 ± 9% of control, n = 5, P = 0.64), whereas application of yohimbine (100 µM), a ₂ receptor antagonist, or propranolol (100 µM), a receptor antagonist, had no effects on NE-induced increases in sIPSC frequency (yohimbine: 168 ± 11% of control, n = 5, P = 0.003; Fig. 2D; propranolol: 158 ± 18% of control, n = 5, P = 0.018; Fig. 2 E) and amplitude (yohimbine: 152 ± 13% of control, n = 5, P = 0.02; propranolol: 164 ± 23% of control, n = 5, P = 0.04). Furthermore, application of phenylephrine (100 µM), a ₁ adrenergic receptor agonist, increased the frequency (177 ± 21% of control, n = 5, P = 0.02; Fig. 2F) and amplitude (163 ± 22% of control, n = 5, P = 0.04) of sIPSCs. Together, these results indicate that the effects of NE on sIPSCs are mediated through activation of ₁ adrenergic receptors in the EC.

NE increases the frequency with no effects on the amplitude of mIPSCs

sIPSCs recorded in the absence of TTX are believed to be action potential- and Ca²⁺-dependent. We next examined the effects of NE on mIPSCs recorded in the presence of TTX (1 µM). Application of NE (100 µM) significantly increased the frequency of mIPSCs (155 ± 8% of control, n = 5, P = 0.002; Fig. 3, A–C) without significantly altering the amplitude of mIPSCs (107 ± 5% of control, n = 5, P = 0.22; Fig. 3D). These results suggest that NE increases presynaptic GABA release with no effects on postsynaptic GABA_A receptors. We compared the kinetics of the averaged mIPSCs before and after the effect of NE reached maximal. NE significantly slowed the decay of mIPSCs (control: 23.3 ± 1.6 ms, NE: 31.8 ± 4.4 ms, n = 5, P = 0.04; Fig. 3E).

View larger version (32K): [in this window] [in a new window]   FIG. 3. NE increases frequency and not amplitude of miniature IPSCs (mIPSCs) and slows decay kinetics of mIPSCs recorded in presence of TTX (1 µM). A: mIPSCs recorded from a neuron before (left) and during (right) application of NE (100 µM). Part of trace is shown in enlarged scale at bottom. B: pooled time-course of mIPSC frequency (n = 5). C: cumulative probability of mIPSC frequency before and during application of NE (n = 5). Note that NE increased mIPSC frequency (reduction in intervals of events). D: cumulative probability of mIPSC amplitude before and during application of NE (n = 5). Note that NE failed to change mIPSC amplitude, suggesting that NE does not modulate postsynaptic GABAA receptors. E: NE slowed decay kinetics of mIPSCs. Individual experiments are indicated by open circles. Mean data are indicated by closed circles. Inset: overlaid traces from the same cell before and after application of NE.

We examined the effects of NE on IPSCs evoked by placing a stimulation electrode locally in the EC to stimulate GABAergic inputs. Because the normal variation of evoked IPSC amplitude was 10% in our recording condition, we defined that synapses showing changes in evoked IPSC amplitude by >15% in response to the application of NE (100 µM) as responsive synapses. Of the 18 synapses examined, 5 synapses exhibited an increase (148 ± 13% of control, n = 5, P = 0.023; Fig. 4, A and D), 6 synapses showed no change (96 ± 3% of control, n = 6, P = 0.21; Fig. 4, B and D), and 7 synapses displayed a decrease (66 ± 4% of control, n = 7, P < 0.001; Fig. 4, C and D) in evoked IPSC amplitude in response to the application of NE (100 µM). The biophysical mechanisms underlying NE-induced heterogeneity of evoked IPSCs may include the selective expression of 1 adrenergic receptors or other relevant release machineries at the stimulated presynaptic terminals and changes in presynaptic release pool at the active zone (see DISCUSSION). Consistent with our results, heterogeneity of NE-induced changes in evoked IPSCs has been observed in different neurons (Bennett et al. 1998; Braga et al. 2004; Hirono and Obata 2006; Madison and Nicoll 1988).

View larger version (29K): [in this window] [in a new window]   FIG. 4. Heterogeneity of NE on evoked IPSCs. A: bath application of NE (100 µM) increased amplitude of evoked IPSCs at an inhibitory synapse in EC. Top: traces averaged from 10 IPSCs taken at time-points indicated in bottom panel. Stimulation artifact was blanked for each trace for clarity. Bottom: time-course of NE-mediated increase in IPSCs. Holding potential was +30 mV. B: application of NE (100 µM) did not obviously change amplitude of evoked IPSCs at an inhibitory synapse in EC. Figure was arranged in the same way as in A. C: application of NE (100 µM) depressed amplitude of evoked IPSCs at an inhibitory synapse in EC. Figure was arranged in the same way as in A and B. D: scatter plot of amplitude of evoked IPSCs from 18 synapses.

mIPSCs recorded in the presence of TTX are independent of action potential, and they are generated by spontaneous vesicle fusion. Whereas the result that NE still increased the frequency of mIPSCs in the presence of TTX suggests that voltage-gated Ca²⁺ channels are unlikely to be responsible for the effects of NE, we still tested the possibility that NE might inhibit resting K⁺ channels to generate membrane depolarization resulting in opening of low-threshold Ca²⁺ channels to increase GABA release. We initially recorded sIPSCs in the absence of TTX and switched to the extracellular solution containing two nonspecific Ca²⁺ channel blockers, Cd²⁺ (100 µM) and Ni²⁺ (100 µM), to block Ca²⁺ influx by voltage-gated Ca²⁺ channels. Application of the Ca²⁺ channel blockers significantly reduced sIPSC frequency (59 ± 10% of control, n = 5, P = 0.016; Fig. 5A) and amplitude (76 ± 7% of control, n = 5, P = 0.02). Subsequent application of TTX (1 µM) after Ca²⁺ channels were blocked did not further significantly reduce the frequency (94 ± 3% of control, n = 5, P = 0.11; Fig. 5A) and amplitude (96 ± 3% of control, n = 5, P = 0.2) of IPSCs, suggesting that voltage-gated Ca²⁺ channels contribute significantly to the generation of sIPSCs. In the presence of Cd²⁺, Ni²⁺, and TTX, application of NE (100 µM) still significantly increased the frequency (163 ± 10% of control, n = 5, P = 0.003; Fig. 5A) without changing the amplitude (102 ± 4% of control, n = 5, P = 0.5) of IPSCs, suggesting that voltage-gated Ca²⁺ channels are unlikely to be involved in NE-induced increases in GABA release. We performed a similar kind of experiment by omitting Ca²⁺ in the extracellular solution. Exclusion of Ca²⁺ in the extracellular solution remarkably reduced IPSC frequency (52 ± 2% of control, n = 6, P < 0.001; Fig. 5B) and amplitude (61 ± 7% of control, n = 6, P = 0.002). Subsequent inclusion of TTX (1 µM) in the extracellular solution failed to change significantly IPSC frequency (100 ± 5% of control, n = 6, P = 0.95; Fig. 5B) and amplitude (98 ± 3% of control, n = 6, P = 0.44). In this recording condition, application of NE (100 µM) still increased the frequency (157 ± 13% of control, n = 6, P = 0.007; Fig. 5B) with no effects on the amplitude (104 ± 2% of control, n = 6, P = 0.14) of IPSCs, further excluding the involvement of voltage-gated Ca²⁺ channels.

View larger version (45K): [in this window] [in a new window]   FIG. 5. NE-mediated facilitation of GABA release is Ca2+-independent. A: application of Ca2+ channel blockers, Cd2+ (100 µM) and Ni2+ (100 µM), failed to block NE-induced increase in mIPSC frequency (n = 7). B: omission of extracellular Ca2+ did not change NE-induced increase in mIPSC frequency (n = 5). C: replacement of extracellular Na+ with the same concentration of N-methyl-D-glucamine (NMDG) failed to alter NE-mediated increase in mIPSC frequency (n = 5). D: application of BAPTA-AM (100 µM) did not block NE-induced increase in mIPSC frequency (n = 5). E: application of thapsigargin (10 µM) failed to block NE-induced increase in mIPSC frequency (n = 5). F: application of ryanodine (100 µM) failed to block NE-induced increase in mIPSC frequency (n = 5).

We finally tested whether an increase in intracellular Ca²⁺ concentration was required for NE-induced increase in GABA release. Inclusion of the membrane-permeable Ca²⁺ chelator, BAPTA-AM (100 µM), in the extracellular solution reduced sIPSC frequency to 54 ± 4% of control (n = 7, P < 0.001; Fig. 5D) and amplitude to 72 ± 4% of control (n = 7, P < 0.001). Subsequent application of TTX (1 µM) did not significantly reduce sIPSC frequency (94 ± 4% of control, n = 7, P = 0.2; Fig. 5D) and amplitude (96 ± 2% of control, n = 7, P = 0.12). In this recording condition, application of NE (100 µM) still significantly increased the frequency (150 ± 8% of control, n = 7, P < 0.001; Fig. 5D), with no effects on the amplitude (101 ± 3% of control, n = 7, P = 0.65) of IPSCs. Furthermore, bath application of thapsigargin (10 µM), a potent inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPases, significantly reduced sIPSC frequency (84 ± 5% of control, n = 5, P = 0.045; Fig. 5E) and amplitude (73 ± 4% of control, n = 5, P = 0.002). In the presence of thapsigargin, application of TTX (1 µM) further reduced sIPSC frequency to 65 ± 5% of control (n = 5, P = 0.002; Fig. 5E) and amplitude to 52 ± 5% of control (n = 5, P = 0.005). After application of NE (100 µM) still significantly increased the frequency (166 ± 8%, n = 5, P = 0.001; Fig. 5E) without altering the amplitude (104 ± 7% of control, n = 5, P = 0.61) of IPSCs. Similarly, bath application of ryanodine (100 µM) reduced sIPSC frequency to 87 ± 3% of control (n = 5, P = 0.02; Fig. 5F) and amplitude to 81 ± 3% of control (n = 5, P = 0.002). In the presence of ryanodine, application of TTX (1 µM) further reduced the frequency (63 ± 11% of control, n = 5, P = 0.03; Fig. 5F) and amplitude (66 ± 8% of control, n = 5, P = 0.013). After application of NE (100 µM) still significantly increased IPSC frequency (157 ± 10% of control, n = 5, P = 0.005; Fig. 5F) with no effects on IPSC amplitude (106 ± 4% of control, n = 5, P = 0.19). Together, these results indicate that the NE-induced increase in GABA release is Ca²⁺-independent.

NE does not modulate the excitability of the interneurons in layer III of the EC

Principal neurons in layer II/III receive GABAergic innervations from local interneurons. We next tested whether NE changes the excitability of the interneurons by recording action potential firing and holding current (at –55 mV) from GABAergic interneurons in layer III of the EC. We identified interneurons by referring to the criteria set by Kumar and Buckmaster (2006). Interneurons in layer III had smaller capacitance (24.6 ± 1.6 pF, n = 10), higher membrane resistance (349 ± 28 M, n = 10), and apparent spike afterhyperpolarization amplitude (–14.7 ± 1.2 mV, n = 10). The identities of the interneurons were further confirmed by ad hoc biocytin staining of the recorded interneurons (Fig. 6D). Application of NE (100 µM) changed neither the frequency of action potentials (96 ± 7% of control, n = 5, P = 0.57; Fig. 6, A and B) nor the holding current recorded at –55 mV in the presence of 1 µM TTX (0.19 ± 0.95 pA, n = 5, P = 0.85; Fig. 6C), suggesting that NE does not influence the excitability of the interneurons.

View larger version (34K): [in this window] [in a new window]   FIG. 6. NE does not change excitability of interneurons in layer III of EC. A: action potentials recorded from an interneuron in layer III of EC in whole cell configuration before (top) and during (bottom) application of NE (100 µM). Note that NE had no effects on action potential firing. B: summarized time-course of action potential firing frequency from 5 cells. C: NE did not change holding current recorded from layer III interneurons (n = 5). D: biocytin-labeled interneuron. E: firing activity recorded from an interneuron in layer III of EC in cell-attached patch before (top) and during (bottom) application of NE (100 µM). Event indicated by arrow was an event plotted in enlarged scale. F: summarized time-course of firing frequency recorded from 7 cells by cell-attached patches.

NE-induced increase in GABA release is independent of PLC, PKC, and tyrosine kinase activities

₁-Adrenergic receptors are G protein–coupled receptors that are coupled to G_q/11 (Hein 2006). Activation of G_q/11 increases the activity of PLC, which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP₂) to produce inositol triphosphate (IP₃) to facilitate intracellular Ca²⁺ release and diacylglycerol to activate PKC. We next tested the roles of this pathway in NE-induced increases in GABA release. Slices were pretreated with U73122 [GenBank] (20 µM), a PLC inhibitor, and the same concentration of U73122 [GenBank] was bath applied. In the presence of U73122 [GenBank] , application of NE (100 µM) still significantly increased the frequency (162 ± 14% of control, n = 7, P = 0.005; Fig. 7A) and amplitude (153 ± 15% of control, n = 7, P = 0.014) of sIPSCs. We also used another PLC inhibitor, edelfosine (Horowitz et al. 2005; Powis et al. 1992). Slices were pretreated with edelfosine (20 µM), and the same concentration of eldefosine was bath applied. In the presence of edelfosine, application of NE (100 µM) still significantly increased the frequency (181 ± 10% of control, n = 5, P = 0.001; Fig. 7B) and amplitude (169 ± 6% of control, n = 5, P < 0.001) of sIPSCs. To test whether U73122 [GenBank] and edelfosine were effective at inhibiting PLC activity, we performed a positive control experiment. Because brain-derived neurotrophic factor (BDNF) has been reported to inhibit GABAergic transmission in CA1 region of the hippocampus through activation of Trk-B and PLC- (Tanaka et al. 1997), we recorded the evoked IPSCs from CA1 pyramidal neurons by placing a stimulation electrode in the stratum pyramidal. Bath application of BDNF (100 ng/ml) significantly inhibited the amplitude of evoked IPSCs (56 ± 9% of control, n = 6, P = 0.005). However, application of the same concentration of BDNF failed to significantly inhibit the amplitude of evoked IPSCs in slices pretreated with U73122 [GenBank] (20 µM, 93 ± 5% of control, n = 6, P = 0.21) or eldefosine (20 µM, 90 ± 5% of control, n = 6, P = 0.12). These results suggest that the activity of PLC is not required for NE-induced increase in GABA release in the EC. Because G protein–coupled receptors are coupled to PLC and among the four isoforms of PLC (PLC1–4), only PLC1 is expressed in the hippocampal formation (Watanabe et al. 1998), we tested the role of PLC in the effects of NE by using PLC1 knockout mice (Deng et al. 2006). Application of NE (100 µM) increased the frequency and amplitude of sIPSC in both wild-type and PLC₁ knockout mice (Fig. 7C). Together, these results indicate that PLC is unlikely to be involved in NE-mediated increase in GABA release. We also tested whether the activity of PKC was necessary for NE-induced increase in GABA release. Application of calphostin C (1 µM), a specific PKC inhibitor, significantly inhibited the basal sIPSC frequency (83 ± 4% of control, n = 5, P = 0.008; Fig. 7D) and amplitude (79 ± 3% of control, n = 5, P = 0.002). In the presence of calphostin C, application of NE (100 µM) still significantly increased sIPSC frequency (170 ± 14% of control, n = 5, P = 0.009; Fig. 7D) and amplitude (151 ± 6% of control, n = 5, P < 0.001). We also used another specific PKC inhibitor, Ro318220. Application of Ro318220 (1 µM) significantly depressed sIPSC frequency (77 ± 6% of control, n = 7, P = 0.007; Fig. 7E) and amplitude (71 ± 3% of control, n = 7, P < 0.001). However, after application of NE (100 µM) still significantly enhanced sIPSC frequency (155 ± 11% of control, n = 7, P = 0.002; Fig. 7E) and amplitude (145 ± 12% of control, n = 7, P = 0.008). Together, these data suggest that the activity of PKC is unnecessary for NE-induced increase in GABA release.

View larger version (41K): [in this window] [in a new window]   FIG. 7. NE-mediated increase in GABAergic transmission does not require functions of phospholipase C (PLC), protein kinase C (PKC), and tyrosine kinase. A: pretreatment of slices with and bath application of U73122 (20 µM), a PLC inhibitor, did not change NE-induced increase in sIPSC frequency (n = 7). B: pretreatment of slices with and bath application of edelfosine (20 µM), another PLC inhibitor, failed to change NE-induced increase in sIPSC frequency (n = 5). C: application of NE (100 µM) increased sIPSC frequency and amplitude to same scale in wild-type (5 slices from 2 mice) and PLC1 knockout mice (7 slices from 2 mice). D: application of a PKC inhibitor, calphostin C (1 µM), did not block NE-induced increase in sIPSC frequency (n = 5). E: application of Ro318220 (1 µM), another PKC inhibitor, failed to change NE-induced increase in sIPSC frequency (n = 7). F: application of genistein (50 µM), a tyrosine kinase inhibitor, failed to change NE-induced increase in sIPSC frequency (n = 6).

NE inhibits the excitability of principal neurons in the EC

If NE increases GABAergic transmission onto the principal neurons, it should reduce the excitability of the principal neurons in the EC. We next recorded from the principal neurons in the EC and tested the effects of NE on action potential firing. Application of NE (100 µM) significantly reduced the frequency of action potentials to 56 ± 5% of control (n = 6, P < 0.001; Fig. 8), showing that NE inhibits neuronal excitability in the EC.

View larger version (27K): [in this window] [in a new window]   FIG. 8. NE reduces excitability of principal neurons in EC. A: action potentials recorded from a principal neuron in EC before and during application of NE (100 µM). B: summarized time-course of action potential firing frequency from 6 cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

Our results showed that NE enhances GABAergic transmission in the EC by increasing presynaptic GABA release without altering postsynaptic GABA_A receptors because NE only increased the frequency without altering the amplitude of mIPSCs, although it increased both the frequency and amplitude of sIPSCs. Our results exclude a role of Ca²⁺ influx through voltage-gated Ca²⁺ channels because application of the Ca²⁺ channel blockers, Cd²⁺ and Ni²⁺, and deprivation of extracellular Ca²⁺ failed to change NE-induced increase in mIPSC frequency. It is also unlikely that NE opens a cationic conductance on the presynaptic membrane to increase GABA release because replacing the extracellular Na⁺ with NMDG did not prevent NE-induced increase in mIPSC frequency. The results that application of BAPTA-AM, thapsigargin, and ryanodine failed to change NE-induced increase in mPSC frequency suggest that Ca²⁺ released from intracellular stores is not required for NE-induced increase in GABA release. Together, these results indicate that NE-mediated facilitation of GABA release is Ca²⁺-independent and mediated by an interaction with GABA release machinery. Consistent with our results, a variety of G protein–coupled receptors including adenosine A1 (Capogna et al. 1996; Scanziani et al. 1992), GABA_B (Capogna et al. 1996; Scanziani et al. 1992), somatostatin (Boehm and Betz 1997), muscarinic (Scanziani et al. 1995), and metabotropic glutamate (Scanziani et al. 1995; Tyler and Lovinger 1995) receptors modulate transmitter release through a direct interaction with the secretory apparatus on the presynaptic terminals.

Dependent on the brain regions, activation of adrenergic receptors modulates GABAergic transmission through at least three distinct ionic mechanisms. First, NE increases the frequency of sIPSCs with no effects on mIPSCs in CA1 pyramidal neurons of the hippocampus (Bergles et al. 1996), the frontal cortex (Kawaguchi and Shindou 1998), and the hypothalamic paraventricular nucleus (Han et al. 2002). The effects of NE in these brain regions are likely caused by NE-induced depression of K⁺ channels in presynaptic GABAergic interneurons (Bergles et al. 1996). This mechanism, however, is not applicable for NE-induced facilitation of GABAergic transmission in the EC because depression of resting membrane K⁺ channels increases action potential firing and Ca²⁺ influx through voltage-gated Ca²⁺ channels, whereas in the EC, NE does not modulate the excitability of interneurons. Second, NE increases the frequency and amplitude of sIPSCs, but only increases the frequency of mIPSCs in sensory motor cortex (Bennett et al. 1998) and in Purkinje cells of the mouse cerebellum (Hirono and Obata 2006). The effects of NE in these brain regions resemble our results, suggesting that they may share the similar ionic mechanism, although the underlying ionic mechanisms of NE in those brain regions have not been determined yet. Based on our results, it is reasonable to speculate that NE may also facilitate GABA release by interacting with the release machinery in those areas. Third, NE increases mIPSC frequency in the accessory olfactory bulb through Ca²⁺ influx mediated by Ca²⁺ channels because NE-induced increase in mIPSC frequency is sensitive to Ca²⁺ channel blockers, Cd²⁺and Ni²⁺ (Araneda and Firestein 2006). Our results suggest that this is not likely the mechanism for NE-mediated increase in GABA release in the EC because application of these Ca²⁺ channel blockers had no effects on NE-induced increase in mIPSC frequency.

We observed that NE slowed the decay of the averaged mIPSCs in the EC. There are two plausible explanations for NE-induced change in mIPSC kinetics. First, our results suggest that the effects of NE are mediated through a direct interaction with the release machinery. If the action site of NE is on the fusion pore, the kinetics of mIPSCs could possibly be altered. Second, NE-induced slowness of mIPSC decay kinetics might be caused by NE-mediated increases in the number of mIPSCs that overlay on the decay phase of previous mIPSCs, resulting in a slowness of the decay kinetics of the averaged mIPSCs.

We also observed that NE generates heterogeneous responses on evoked IPSCs. Application of NE increased, did not change, or decreased the amplitude of evoked IPSCs at individual synapses in the EC. Consistent with our results, NE has been shown to depress evoked IPSCs in the hippocampus (Madison and Nicoll 1988), but increase evoked IPSCs in Purkinje cells of mouse cerebellum (Hirono and Obata 2006) and in basolateral amygdala neurons (Braga et al. 2004). NE-mediated increase, decrease, and no change in evoked IPSCs have been observed in rat sensorimotorcortex (Bennett et al. 1998). Whereas the exact mechanisms underlying the heterogeneous effects of NE on the evoked IPSCs remain to be determined, we propose three possible mechanisms to explain our results. First, NE-mediated increase in evoked IPSC amplitude at some synapses may reflect the true effects of NE, i.e., enhancement of GABA release. At these synapses, the presynaptic terminals of the stimulated fibers are likely to express 1 adrenergic receptors and other required machineries. Second, different from sIPSCs and mIPSCs, which are likely from many different synapses onto the recorded neurons, evoked IPSCs are generated by a few fibers that are stimulated exogenously. If the stimulated fibers do not express 1 adrenergic receptors or other release machineries required by NE, application of NE would generate no responses. This may explain the results that NE had no effects on the amplitude of evoked IPSCs at some synapses. Third, NE-induced increase in spontaneous GABA release at many release sites resembles a condition named asynchronous release. NE-induced increases in asynchronous release may have reduced the size of the release pool at the active zone resulting in a reduction in evoked IPSCs (synchronous release). Whereas the heterogeneous effects of NE on evoked IPSCs may be generated by distinct biophysical mechanisms, our result that NE increased sIPSC frequency and amplitude in every cell examined (Fig. 1) suggest that NE makes considerable contribution to the inhibition in the EC because sIPSCs represent a more natural transmission in vivo.

Our results show that NE increases GABA release through activation of ₁ adrenergic receptors without the requirement of ₂ or receptors, consistent with the results from other synapses (Araneda and Firestein 2006; Bennett et al. 1998; Bergles et al. 1996; Braga et al. 2004; Han et al. 2002; Hirono and Obata 2006). ₁ receptors are coupled to G_q/11, resulting in activation of the PLC pathway. We showed that the function of PLC is unnecessary for NE-mediated facilitation of GABA release because application of NE still increased GABA release in the presence of two PLC inhibitors (U73122 [GenBank] and edelfosine) and in PLC₁ knockout mice. Because a general caveat for the experiments of knockout animals is that the knockout animals can potentially produce compensatory signals, we cannot rule out this possibility for the experiments involving PLC₁ knockout mice, although PLC1 is the type of PLC expressed in the hippocampal formation (Watanabe et al. 1998) and there has been no report in the literature suggesting that the PLC1 knockout mice generate other PLC isoforms to compensate the deleted PLC1. Our results do not support any roles of the two downstream targets of PLC (IP₃-mediated intracellular Ca²⁺ release and PKC) in NE-mediated increase in GABA release because application of BAPTA-AM to chelate intracellular Ca²⁺ and two PKC inhibitors (calphostin C and Ro318220) failed to change NE-mediated GABA release. Together, these results suggest a mode in which G proteins activated by ₁ receptors directly interact with the release machinery to facilitate GABA release. Consistent with this notion, G released by activation of G protein–coupled serotonin receptors modulates transmitter release through direct interaction with exocytotic fusion machinery (Blackmer et al. 2001, 2005; Gerachshenko et al. 2005; Photowala et al. 2006).

In addition to these findings that NE facilitates GABAergic transmission through ₁ receptors, NE also inhibits glutamatergic transmission through ₂ receptors in the EC (Pralong and Magistretti 1994, 1995). The inhibitory effects of ₂ receptors on excitatory synaptic transmission in the EC may explain NE-mediated antiepileptic actions in a bicuculline-induced seizure model (Stoop et al. 2000). Nonetheless, NE has been reported to block low Mg²⁺-induced epileptiform activity through ₁ receptors in the EC (Stanton et al. 1987). Our results can explain the discrepancy of these results, because NE-induced facilitation of GABAergic transmission through ₁ receptors was overwhelmed in a bicuculline-induced seizure model, whereas it was functional in a low Mg²⁺-induced epileptic model. Therefore NE-mediated facilitation of GABAergic transmission is likely to be an important player in NE-induced inhibition of epilepsy in the EC.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institutes of Health, National Center for Research Resources Grant 5P20RR-017699-02. H.-S. Shin is supported by a National Honor Scientist grant from the Ministry of Science and Technology, Republic of Korea, and a Center-of-Excellence grant from Korea Institute of Science and Technology.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Lei, Dept. of Pharmacology, Physiology and Therapeutics, School of Medicine and Health Sciences, Univ. of North Dakota, Grand Forks, ND 58203 (E-mail: slei{at}medicine.nodak.edu)

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