Korean J Physiol Pharmacol.  2019 Sep;23(5):317-328. 10.4196/kjpp.2019.23.5.317.

Layer-specific cholinergic modulation of synaptic transmission in layer 2/3 pyramidal neurons of rat visual cortex

• Cho KH
• Lee SY
• Joo K
• Rhie DJ

• KMID: 2455808
• DOI: http://doi.org/10.4196/kjpp.2019.23.5.317

Abstract

It is known that top-down associative inputs terminate on distal apical dendrites in layer 1 while bottom-up sensory inputs terminate on perisomatic dendrites of layer 2/3 pyramidal neurons (L2/3 PyNs) in primary sensory cortex. Since studies on synaptic transmission in layer 1 are sparse, we investigated the basic properties and cholinergic modulation of synaptic transmission in layer 1 and compared them to those in perisomatic dendrites of L2/3 PyNs of rat primary visual cortex. Using extracellular stimulations of layer 1 and layer 4, we evoked excitatory postsynaptic current/potential in synapses in distal apical dendrites (L1-EPSC/L1-EPSP) and those in perisomatic dendrites (L4-EPSC/L4-EPSP), respectively. Kinetics of L1-EPSC was slower than that of L4-EPSC. L1-EPSC showed presynaptic depression while L4-EPSC was facilitating. In contrast, inhibitory postsynaptic currents showed similar paired-pulse ratio between layer 1 and layer 4 stimulations with depression only at 100 Hz. Cholinergic stimulation induced presynaptic depression by activating muscarinic receptors in excitatory and inhibitory synapses to similar extents in both inputs. However, nicotinic stimulation enhanced excitatory synaptic transmission by ~20% in L4-EPSC. Rectification index of AMPA receptors and AMPA/NMDA ratio were similar between synapses in distal apical and perisomatic dendrites. These results provide basic properties and cholinergic modulation of synaptic transmission between distal apical and perisomatic dendrites in L2/3 PyNs of the visual cortex, which might be important for controlling information processing balance depending on attentional state.

Keyword

Cholinergic modulation; Layer 2/3; Layer-specific; Synaptic transmission; Visual cortex

MeSH Terms

Animals
Automatic Data Processing
Dendrites
Depression
Inhibitory Postsynaptic Potentials
Kinetics
Pyramidal Cells*
Rats*
Receptors, AMPA
Receptors, Muscarinic
Synapses
Synaptic Transmission*
Visual Cortex*
Receptors, AMPA
Receptors, Muscarinic

Figure

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  Fig. 1 Independency of layer 1 (L1) and L4 stimulations. (A) Experimental configuration (5x objective image) showing positions of glass and tungsten electrodes at L1 and L4, respectively. A patch pipet recording a layer 2/3 pyramidal neuron was indicated as P. Image of biocytin-filled cell is overlaid. Scale = 200 µm. (B) Excitatory postsynaptic currents (EPSCs) evoked by combined stimulation of L1 and L4 (L1 + L4) were compared with the arithmetic sum of each EPSC (right panel, n = 5). (C) Cross paired-pulse stimulation of L1 and L4 stimulations indicated the independency of these two inputs. Relative EPSC amplitude was obtained from peak amplitude of the second EPSC of cross paired-pulse stimulation (L1–L4, L4–L1) versus peak amplitude of the first EPSC of paired-pulse stimulation (L1–L1, L4–L4) (n = 6).

• 

  Fig. 2 Kinetics of excitatory postsynaptic current (EPSC) and excitatory postsynaptic potential (EPSP) evoked by layer 1 (L1) and L4 stimulations. (A) EPSCs (upper traces) and EPSPs (lower traces) were recorded with stimulations of L1 (thin trace) and L4 (thick trace). EPSC(P) traces were normalized to the peak. (B) Summary plots of peak time and decay time constant (tau) of EPSC (n = 11) and EPSP (n = 9) evoked by stimulations of L1 (open bar) and L4 (closed bar). *p < 0.05, **p < 0.01, ***p < 0.001 compared to L1.

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  Fig. 3 Short-term kinetics of excitatory postsynaptic current (EPSC) evoked by layer 1 (L1) and L4 stimulations. (A) Left panel shows representative currents evoked by stimulations of L1 (dotted line) and L4 (solid line) recorded at holding potential of −80 mV. Middle and right panels show representative currents measured at holding potential of −80 mV and 0 mV, respectively, in the presence of 6,7-dinitroquinoxaline-2,3-dione disodium salt (DNQX) (20 µM) and D-(−)-2-Amino-5-phosphonopentanoic acid (D-AP5) (50 µM) in bath solution (n = 3). (B) Representative traces from paired-pulse stimulations with interstimulus interval (ISI) of 10 to 200 ms, evoked by L1 stimulation (upper traces) and L4 stimulation (lower traces). EPSC traces were normalized to the first peak. (C) Summary of paired-pulse ratio (PPR) with stimulations of L1 and L4 (n = 13). *p < 0.05, **p < 0.01, ***p < 0.001 compared to L4.

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  Fig. 4 Cholinergic modulation of excitatory postsynaptic current (EPSC) evoked by layer 1 (L1) and L4 stimulations. EPSCs were evoked by stimulations of L1 (A) and L4 (B) with bath application of cholinergic agonists at various concentrations. Representative traces of EPSC at various concentrations (µM) of muscarine and nicotine are shown in the right panel. All drugs were applied into bath solution and effects were analyzed at 5 min after application of the drugs (n = 8–14). EPSCs were decreased by non-specific cholinergic agonist carbachol and muscarinic receptor agonist muscarine in a concentration-dependent manner. However, nicotinic receptor agonist nicotine increased L4-EPSC at concentrations lower than 1 µM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with normal bath solution.

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  Fig. 5 Presynaptic effect of cholinergic modulation on excitatory postsynaptic current (EPSC). (A) Experimental configuration showing positions of glass puff-pipettes (layer 1 [L1] and L2/3) and a recording patch pipet with an L2/3 pyramidal neuron. (B) Inward currents were evoked by puff application of glutamate (0.3–3 mM) in L1 and L2/3 (perisomatic area). Puffs were applied every 20 sec at 5 psi for 5–30 ms using a glass pipet (bars on traces). Muscarine (10 µM, 5 min) was applied into the bath solution. Upper traces show representative average EPSCs taken at indicated time period (2 min). (C) Summary plot for effects of various cholinergic agonists (10 µM) on amplitude of EPSCs evoked by puff application of glutamate (n = 5) recorded at the end of application (CCh, carbachol; Mus, muscarine; Nic, nicotine). (D) Representative traces of muscarine (10 µM) application on miniature EPSCs (mEPSCs) recorded in the presence of tetrodotoxin (1 µM). (E) Summary plots of muscarine effects on mEPSCs amplitude and frequency (n = 11). ***p < 0.001 tested with ANOVA.

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  Fig. 6 Short-term kinetics of inhibitory postsynaptic current (IPSC) evoked by layer 1 (L1) and L4 stimulations. IPSCs were evoked by paired-pulse stimulation with various interstimulus interval (ISI) with L1 and L4 stimulations in the presence of 6,7-dinitroquinoxaline-2,3-dione disodium salt (DNQX) (20 µM) and D-(−)-2-Amino-5-phosphonopentanoic acid (D-AP5) (50 µM). (A) Representative IPSCs evoked by paired-pulse stimulations with ISI of 10 to 200 ms, evoked by L1 stimulation (upper traces) and L4 stimulation (lower traces). IPSC traces were normalized to the first peak. (B) Summary plot shows paired-pulse ratio (PPR) with L1 stimulation (n = 14) and L4 stimulation (n = 16). *p < 0.05, **p < 0.01 tested with ANOVA.

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  Fig. 7 Cholinergic modulation of inhibitory postsynaptic currents (IPSCs) evoked by layer 1 (L1) and L4 stimulations. IPSCs were evoked by stimulating L1 (A) and L4 (B) in the presence of various cholinergic agonists. Representative traces of inhibitory postsynaptic current (IPSC) at various concentrations (µM) of muscarine and nicotine were shown in the right panel. All drugs were applied in the bath solution and their effects were analyzed at 5 min after application. IPSCs were declined by carbachol (n = 10) and muscarine (n = 7) in a concentration-dependent manner. Nicotine had no effects on L1 or L4 stimulation (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001 compared with normal bath solution.

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  Fig. 8 Properties of glutamatergic synapses activated by layer 1 (L1) and L4 stimulations. (A) Excitatory postsynaptic currents (EPSCs) were evoked by L1 stimulation (left panel) or L4 stimulation (middle panel) at holding potential of either −80 or +40 mV. Amplitude of AMPA receptor-mediated EPSC was analyzed at peak of synaptic response recorded at −80 mV (dotted line). Amplitude of NMDA receptor-mediated EPSC was analyzed 50 ms after stimulus onset at +40 mV (solid line). Ratio of AMPA/NMDA receptor-mediated EPSCs was obtained from these values (right panel, n = 5–7). (B) AMPA receptor-mediated EPSCs were measured at various membrane potentials. Current-voltage (I–V) curve of L1 (left panels) and L4 stimulation (right panels) are plotted. Rectification index of AMPA receptor-mediated EPSCs was calculated using the following equation: I+40 / (40 − Erev) / I−40 (Erev + 40). Reversal potential for AMPA receptor mediated-EPSC (Erev) was obtained from its I–V relationship.

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