Multiple receptors coupled to phospholipase C gate long-term depression in visual cortex

Abstract

Long-term depression (LTD) in sensory cortices depends on the activation of NMDA receptors. Here, we report that in visual cortical slices, the induction of LTD (but not long-term potentiation) also requires the activation of receptors coupled to the phospholipase C (PLC) pathway. Using immunolesions in combination with agonists and antagonists, we selectively manipulated the activation of alpha1 adrenergic, M1 muscarinic, and mGluR5 glutamatergic receptors. Inactivation of these PLC-coupled receptors prevents the induction of LTD, but only when the three receptors were inactivated together. LTD is fully restored by activating any one of them or by supplying intracellular D-myo-inositol-1,4,5-triphosphate (IP3). LTD was also impaired by intracellular application of PLC or IP3 receptor blockers, and it was absent in mice lacking PLCbeta1, the predominant PLC isoform in the forebrain. We propose that visual cortical LTD requires a minimum of PLC activity that can be supplied independently by at least three neurotransmitter systems. This essential requirement places PLC-linked receptors in a unique position to control the induction of LTD and provides a mechanism for gating visual cortical plasticity via extra-retinal inputs in the intact organism.

Figure 1.

Antagonists of PLC-coupled receptors block the induction of LTD. A, Bath application of 10 μm MPEP, 5 μm atropine, and 10 μm urapidil blocks the induction of LTD. The graph shows the effects of LFS (1 Hz, 15 min) on the FP amplitude in normal ACSF (open circles) or in the presence of the drugs (filled circles). The magnitude of LTD at the end of each individual experiment is indicated on the right of the graph. Examples of responses recorded 1 min before (thin traces) or 60 min after (thick traces) LFS are shown above. B, LTD (expressed as percentage of decrease from baseline) is not blocked when only two antagonists are present in the bath. Asterisks denote statistical significance from control. C, Effects of bath-applied antagonists (MUA) on baseline synaptic responses (top, open symbols) and after the induction of LTD (bottom, filled symbols). D, Application of antagonists (MUA) does not affect the FP amplitude during LFS (1 Hz, 900 pulses). Each data point (open circles, control; filled circles, MUA) is an average of four consecutive responses. E, Application of antagonists does not affect NMDAR responses. Left, Representative synaptic currents recorded at -80 and +40 mV with and without the antagonist mixture (MUA). Right, NMDAR/AMPAR ratios and AMPAR amplitude in the presence and absence of MUA. Calibration: 100 ms, 0.2 nA. F, Application of antagonists (MUA) do not affect the isolated NMDA responses (recorded at -40 mV; see Materials and Methods) during conditioning stimulation (200 pulses, 1 Hz). A, Atropine (5 μm); M, MPEP (10 μm); U, urapidil (10 μm); L, LY341495 (100 μm); Pr, pirenzepine (10 μm); Pz, prazosin (10 μm); Cont, control. Error bars represent SEM.

Figure 2.

Antagonists against PLC-coupled receptors block pairing-induced LTD and promote pairing-induced LTP. A-C, Changes in the EPSP slope induced by pairing in normal ACSF (open circles) in the presence of the drugs (MUA; filled circles) are shown. The pairings were 1 Hz, 200 pulses plus depolarization to -40 mV (A), to -20 mV (B), and to 0 mV (C). C, Control. D, Blockade of PLC-coupled receptors causes an upward shift in the voltage dependence of pairing-induced LTD. Asterisks denote statistical significance from control. Individual experiments are shown on the right of each graph. Superimposed traces are average of four consecutive responses recorded 1 min before (thin traces) or 30 min after (thick traces) pairing. Calibration: 10 ms, 5 mV.

Figure 3.

Immunohistochemical confirmation of cholinergic and noradrengic lesions. A, B, Photomicrographs of the visual cortex show abundant DBH-immunopositive noradrenergic fibers in a control brain (A) and sparse density in a lesioned brain (B). C, D, Photomicrographs of the visual cortex show AChE staining in a control brain (C), which is substantially depleted in an immunolesioned brain (D). Scale bars: A, B, 100 μm; C, D, 200 μm. Immunostaining for the subcortical cell bodies of these projection systems also showed marked removal in lesioned brains (data not shown).

Figure 4.

LTD in immunolesioned slices. A, Bath application of the mGluR5 antagonist MPEP blocks LTD in slices from lesioned animals (192 IgG-saporin and DBH-saporin). The graph shows LTD induced in slices prepared from sham-operated animals (circles) and lesioned animals (triangles) superfused with normal ACSF (open symbols) or with 10 μm MPEP (filled symbols). B, Bath application of the mGluR5 antagonist MPEP also blocks LTD in slices from animals lesioned with 192 IgG-saporin and reserpine. C, Bath application of the M1 agonist McN or the α1 agonist methoxamine rescues LTD in slices from lesioned animals treated with MPEP. The graph shows LTD induced in slices from lesioned animals in the presence of 10 μm MPEP (filled triangles) and in the presence of 10 μm MPEP plus 5 mm McN (open triangles) or 40 μm methoxamine (filled triangles). Results from individual experiments (as percentage of LTD at 60 min after LFS) are shown on the right; representative recordings (before and after LFS) are shown at the top of each graph. S, Sham; M, MPEP; L, LY341495; Metx, methoxamine.

Figure 5.

Type II mGluRs are not essential for NMDAR-dependent LTD in the visual cortex. The average effects of LFS on the FP amplitude in slices superfused with normal ACSF (C; open circles), in the presence of 100 μm MCCG (MC; filled circles), 100 μm LY341495 (LY; filled triangles), and 100 μm MCCG plus 10 μm atropine and 10 μm urapidil (MAU; open triangles) are shown. The results (as percentage of LTD 60 min after LFS) of all individual experiments are shown to the right of the graph. Representative experiments are shown at the top of the graphs.

Figure 6.

Pairing-induced LTD requires postsynaptic PLC. A, Impaired LTD in PLCβ1 knock-out mice. The averaged effects of pairing in cells from homozygotes (hom and Homo; filled circles), heterozygotes (het and Hetero; filled triangles), and their age-matched wild type (wt and Wt; open circles) and littermates. B, Postsynaptic application of the PLC blocker U73122 impairs LTD. The averaged effects of pairing in cells recorded with electrodes filled with control 0.001% DMSO solution (C; open circles), 10 μm U73122 (U.. .2; filled circles), or the inactive isomer 10 μm U73343 (U.. .3; filled triangles) are shown. Results from individual experiments are shown on the right. Traces are averages of four consecutive responses recorded 1 min before (thin traces) and 40 min after (thick traces) pairing.

Figure 7.

U73122 blocks the induction, not the expression, of LTD. A, Reversibility of LTD block. The effects of LFS applied in the presence of 10 μm U73122 (shaded area; left) and 30-40 min after washing out the drug (right) are shown. B, Average effects of the FP amplitude of U73122 (shaded area) applied in naive slices (top, open circles) and 20 min after the induction of LTD (bottom, filled circles). Representative traces in A and B are averages of four consecutive responses recorded before (thin traces) and 30-60 min after (thick traces) LFS.

Figure 8.

LTD induction requires activation of IP₃ receptors. A, Bath application of the PKC blocker GF109203X (GFX; 0.5 μm) does not affect LFS-induced LTD. Open circles, Control; filled circles, GFX. B, Intracellular application of the IP₃ receptor blocker 2-APB (0.5 μm) blocks pairing-induced LTD of the EPSCs (recorded under voltage clamp at -80 mV). Open circles, Control DMSO; filled circles, 2-APB, DMSO control. C, Summary of the effects on LTD of PKC blockers [GFX and staurosporine (Stau.)] and IP₃ receptor blockers [2-APB and heparin (Hep.)]. LTD is expressed as a decrease from baseline. Asterisks indicate significant difference (p < 0.05) from control. D, Postsynaptic application of IP₃ relieves the block of pairing-induced LTD by antagonists against PLC-coupled receptors (MUA: 10 μm MPEP, 5 μm atropine, and 10 μm urapidil). The averaged effects of pairing in cells recorded in control ACSF (Con; open circles), in MUA (filled circles), or with MUA with IP₃ (200 μm) in the pipette (filled triangles) are shown. Results from individual experiments are shown on the right of each panel. Traces are averages of four consecutive responses recorded 1 min before (thin traces) and 40 min after (thick traces) attempting LTD. Con and Cont., Control.

Figure 9.

Antagonists of PLC-coupled receptors reduce the induction of LTD in the temporal cortex and CA1. A, B, Bath application of 10 μm MPEP, 5 μm atropine, and 10 μm prazosin reduces the induction of LTD of the layer IV-III pathway in the temporal cortex (A) and the Schaffer collateral→CA1 pathway in the hippocampus (B). Individual experiments are shown on the right of each graph. Traces are examples of responses recorded 1 min before (thin traces) or 60 min after (thick traces) LFS. C, LTD in CA1 is not affected when only two antagonists are present in the bath. C, Control; A, atropine (5 μm); M, MPEP (10 μm); P, prazosin (10 μm); S, sulpiride (10 μm); K, ketanserin (10 μm). Asterisks denote statistical significance from control.