Korean J Physiol Pharmacol.  2020 Nov;24(6):517-527. 10.4196/kjpp.2020.24.6.517.

Layer-specific serotonergic induction of long-term depression in the prefrontal cortex of rats

• Shin D
• Cho KH
• Joo K
• Rhie DJ

• KMID: 2507734
• DOI: http://doi.org/10.4196/kjpp.2020.24.6.517

Abstract

Layer 2/3 pyramidal neurons (L2/3 PyNs) of the cortex extend their basal dendrites near the soma and as apical dendritic tufts in layer 1, which mainly receive feedforward and feedback inputs, respectively. It is suggested that neuromodulators such as serotonin and acetylcholine may regulate the information flow between brain structures depending on the brain state. However, little is known about the dendritic compartment-specific induction of synaptic transmission in single PyNs. Here, we studied layer-specific serotonergic and cholinergic induction of long-term synaptic plasticity in L2/3 PyNs of the agranular insular cortex, a lateral component of the orbitofrontal cortex. Using FM1-43 dye unloading, we verified that local electrical stimulation to layers 1 (L1) and 3 (L3) activated axon terminals mostly located in L1 and perisomatic area (L2/3). Independent and AMPA receptor-mediated excitatory postsynaptic potential was evoked by local electrical stimulation of either L1 or L3. Application of serotonin (5-HT, 10 μM) induced activity-dependent longterm depression (LTD) in L2/3 but not in L1 inputs. LTD induced by 5-HT was blocked by the 5-HT₂ receptor antagonist ketanserin, an NMDA receptor antagonist and by intracellular Ca²⁺ chelation. The 5-HT₂ receptor agonist α-me-5-HT mimicked the LTD induced by 5-HT. However, the application of carbachol induced muscarinic receptor-dependent LTD in both inputs. The differential layer-specific induction of LTD by neuromodulators might play an important role in information processing mechanism of the prefrontal cortex.

Keyword

Cholinergic; Layer 2/3; Orbitofrontal cortex; Pyramidal cell; Synaptic transmission

Figure

• 

  Fig. 1 Layer-specific activation of Layer 2/3 pyramidal neurons with local electrical stimulation. Experimental configuration (10× objective image) showing numbered regions of interest (ROIs) with stimulating electrodes marked with E1 for layer 1 (L1) stimulation and E3 for layer 3 (L3) stimulation. Scale = 100 μm. (B) Fluorescence changes in ROIs with time (5-sec interval). E1 and E3 were applied at 2 Hz (bars). Data are from a representative experiment (n = 3). (C) Spatial profiles of relative fluorescence changes against distance from the pia. The relative decrease in fluorescence to the maximal decrease was plotted. *p < 0.05, **p < 0.01, ***p < 0.001 compared with L1 or L3 stimulation. (D) Activation of non-overlapping synapses. Upper traces show representative EPSPs evoked by E1 and E3. Middle traces show representative EPSP evoked by combining E1 with E3 and arithmetic sum of corresponding E1-EPSP and E3-EPSP. Lower panel shows individual data of the amplitude of arithmetic sum relative to the amplitude of EPSPs evoked by combined E1 and E3 (n = 12). EPSP, excitatory postsynaptic potential.

• 

  Fig. 2 AMPA receptor-mediated synaptic transmission in Layer 2/3 (L2/3) pyramidal neurons of orbitofrontal cortex. (A) Representative EPSPs evoked by L1 and L3 stimulation (E1 and E3, respectively). The average EPSP waveforms recorded during 1 min at the time indicated by numbered arrows in B. (B) Time course of EPSP amplitude with the application of blockers. The amplitude of first EPSPs evoked by paired-pulse stimulation in the presence and absence of D-AP5 (50 μM) and/or DNQX (20 μM) in bath solution. (C) Summary plot of EPSP amplitude by E1 (n = 6) and E3 (n = 5). ***p < 0.001 compared with baseline. EPSP, excitatory postsynaptic potential; D-AP5, d-(-)-2-amino-5-phosphonopentanoic acid; DNQX, 6,7-dinitroquinoxaline-2,3-dione disodium salt; ACSF, artificial cerebrospinal fluid.

• 

  Fig. 3 Serotonergic induction of long-term depression (LTD) in the Layer 2/3 (L2/3) pyramidal neurons of orbitofrontal cortex. (A) Time course of average EPSP amplitude evoked by L1 and L3 stimulation. Serotonin (5-HT; 10 μM, 10 min) was applied in bath solution (closed symbols: n = 9). In some experiments electrical stimulation was omitted during the application of 5-HT and until 10 min after washout (open symbols: n = 6). Insets show average EPSPs taken at the time indicated from a representative experiment. (B) Left panels show individual data and averages (thick solid lines). Right panels show the change in paired-pulse ratio (PPR) of closed symbols of the left panels. **p < 0.01 compared with the baseline and ##p < 0.01. (C) Concentration response plot of EPSP amplitude with L3 stimulation. The curve was fitted by sigmoid function (EC50 = 3.57 μM). ***p < 0.001 compared with the normal ACSF. EPSP, excitatory postsynaptic potential; ACSF, artificial cerebrospinal fluid.

• 

  Fig. 4 5-HT2 receptor mediates the effect of 5-HT induction of long-term depression (LTD). Summary plots of average EPSP amplitude with L1 (A) and L3 (B) stimulation of Layer 2/3 (L2/3) pyramidal neurons of orbitofrontal cortex. 5-HT antagonists were applied into the bath throughout the experiments. 5-HT (n = 9), α-me-5-HT (n = 6), 8-OH-DPAT (n = 6), mCPBG (n = 7), α-me-5-HT + ketanserin (n = 6), ketanserin (n = 7), NAN-190 (n = 5), and ondansetron (n = 6). 5-HT, serotonin; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin hydrobromide; mCPBG, 1-(m-chlorophenyl) biguanide hydrochloride; α-me-5-HT, α-methyl-5-hydroxytryptamine; EPSP, excitatory postsynaptic potential. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the baseline.

• 

  Fig. 5 Serotonergic long-term depression (LTD) is dependent on intracellular Ca2+ and NMDA receptors. (A) Time course of average EPSP amplitude in the presence of intracellular BAPTA (10 mM) with L1 stimulation (open circle, n = 6) and L3 stimulation (closed circle, n = 6). (B) Time course of average EPSP amplitude with bath application of D-AP5 (50 μM) evoked by L1 stimulation (open circle, n = 5) and L3 stimulation (closed circle, n = 6). (C) Time course of average EPSP amplitude with bath application of AM251 (5 μM) evoked by L1 stimulation (open circle, n = 5) and L3 stimulation (closed circle, n = 6). Insets show average EPSPs taken at the time indicated from a representative experiment. Right panels show EPSP amplitude in individual experiments and the average (solid line). EPSP, excitatory postsynaptic potential; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid; D-AP5, d-(-)-2-amino-5-phosphonopentanoic acid; AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide. **p < 0.01 compared with baseline (pre).

• 

  Fig. 6 Cholinergic induction of long-term depression (LTD). (A)Time course of average EPSP amplitude evoked by L1 and L3 stimulation. Carbachol (CCh, 10 μM) was applied in bath solution (closed circle: n = 9, closed triangle: n = 6). Atropine (10 μM) was applied to the bath solution throughout the experiment (open circle: n = 7, open triangle: n = 5). Insets show average EPSPs taken at the time indicated from a representative experiment. (B) Left panels show individual data and averages (thick solid lines). Right panels show the change in paired-pulse ratio (PPR) of closed symbols of the left panels. EPSP, excitatory postsynaptic potential. **p < 0.01 compared with the baseline, #p < 0.05, ##p < 0.01.

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