Circuit-selective properties of glutamatergic inputs to the rat prelimbic cortex and their alterations in neuropathic pain

Abstract

Functional deactivation of the prefrontal cortex (PFC) is a critical step in the neuropathic pain phenotype. We performed optogenetic circuit dissection to study the properties of ventral hippocampal (vHipp) and thalamic (MDTh) inputs to L5 pyramidal cells in acute mPFC slices and to test whether alterations in these inputs contribute to mPFC deactivation in neuropathic pain. We found that: (1) both the vHipp and MDTh inputs elicit monosynaptic excitatory and polysynaptic inhibitory currents. (2) The strength of the excitatory MDTh input is uniform, while the vHipp input becomes progressively stronger along the dorsal-ventral axis. (3) Synaptic current kinetics suggests that the MDTh inputs contact distal, while the vHipp inputs contact proximal dendritic sections. (4) The longer delay of inhibitory currents in response to vHipp compared to MDTh inputs suggests that they are activated by feedback and feed-forward circuitries, respectively. (5) One week after a peripheral neuropathic injury, both glutamatergic inputs are modified: MDTh responses are smaller, without evidence of presynaptic changes, while the probability of release at vHipp-mPFC synapses becomes lower, without significant change in current amplitude. Thus, dysregulation of both these inputs likely contributes to the mPFC deactivation in neuropathic pain and may impair PFC-dependent cognitive tasks.

Keywords: Channelrhodopsin; Connectivity; Hippocampus; Pyramidal cell; SNI; Thalamus.

Figure 1. Optogenetic dissection of MDTh to mPFC and vHipp to mPFC circuits

A, Experimental design and timeline. B, Allodynic thresholds for the left (injured) paw reveal strong tactile allodynia in SNI compared to sham (SNI, n=15, 1.83±0.72; sham, n=17, 10.73±2.36; Student’s t-test, two-tailed, p=0.002). C, Sites of viral microinjections in MDTh (top) and vHipp (bottom). D, Locations of recorded neurons in the prelimbic mPFC (between 1500 and 3400 μm from the apex) in layer 5 (between 550 and 1000 μm from the pia.

Figure 2. Differential effects of MDTh or vHipp input stimulation on layer 5 pyramidal neurons in the prelimbic mPFC

A, Excitatory responses to a 1 ms blue light activation of MDTh (green) and vHipp (purple) afferents. Holding potential: −70 mV; internal solution was potassium based. B, Summary of response amplitudes in sham animals (MDTh, n=24; vHipp, n=29); vHipp responses are smaller than MDTh responses (MDTh, n=24, 223±44 pA, vHipp, n=29, 102±22 pA, Wilcoxon rank-sum test, two-tailed, Z=2.48, p=0.013). C,D, Amplitude of MDTh (C) and vHipp responses (D), vs. cell location on the dorsal-ventral axis. Amplitudes of vHipp responses show a significant positive correlation with distance from the apex (r²=0.21, p=0.004).

Figure 3. MDTh and vHipp responses are largely monosynaptic

Analysis of voltage clamp recordings from PL mPFC layer 5 pyramidal neurons in slices from control (sham operated) rats; Holding potential: −70 mV. A, Distribution of the delay to onset in response to stimulation of MDTh (n=19, 2.38±0.56 ms) and vHipp inputs (n=24, 2.12±0.19 ms). B, Distribution of response jitter for MDTh responses (n=19, 2.02±0.42 ms) and vHipp responses (n=24, 1.81±0.22 ms). C, Representative traces in slices from sham animals with MDTh or vHipp AAV injections (holding at −70 mV, K-gluconate intrapipette solution). TTX and 4-AP were added to the bath solution to isolate the monosynaptic components.

Figure 4. MDTh and vHipp glutamatergic responses have different kinetics

A, representative excitatory current responses (holding at −70 mV, K-gluconate intra-pipette solution) to stimulation of MDTh (green) and vHipp (purple) projections in PL mPFC slices of control (sham) rats. Exponential fits of the rise and decay are shown in red. B, The 20-80% rise time was significantly longer for the MDTh input (MDTh, n=15, 3.61±0.49 ms; vHipp, n=22, 2.15±0.21 ms; Wilcoxon rank-sum test, two-tailed, Z=2.89, p=0.004). C, the decay time constant was also significantly longer in response to MDTh compared to vHipp input stimulation (MDTh n=15, 21.97±1.93 ms; vHipp, n=22, 12.69±0.83 ms; Wilcoxon rank-sum test, two-tailed, Z=3.85, p=0.0001).

Figure 5. MDTh input stimulation elicits larger, slower responses and recruits more local inhibition compared to vHipp

A, Representative excitatory (inward; holding at −70 mV) and inhibitory (outward; holding at 0 mV) current in response to stimulation of MDTh (green) and vHipp (purple) projections. For these recordings, the intrapipette solution contained Cs-methanesulfonate and 5 mM QX-314 was used to improve the voltage clamp. B, The excitatory component of MDTh afferent input is larger than the vHipp inputs (MDTh, n=8, 798±97 pA; vHipp, n=11, 169±43 pA; Wilcoxon rank-sum test, two-tailed, Z=3.51, p=0.0004). C, The inhibitory component of the MDTh input is also larger (MDTh, n=8, 1765±316 pA, vHipp, n=9, 127±196 pA; Wilcoxon rank-sum test, two-tailed, Z=3.44, p=0.0006). D, The inhibitory/excitatory current ratio is larger for the MDTh input, suggesting that it recruits more inhibitory circuitry than the vHipp input (MDth, n=8, 2.28±0.35; vHipp, n=9, 0.61±0.26; Wilcoxon rank-sum test, two-tailed, Z=2.86, p=0.004). E-G, The kinetics of the inhibitory component also differs between the two inputs. Responses to MDTh input stimulation have shorter delay (MDTh, n=8, 3.23±0.23 ms, vHipp, n=5, 6.22±0.96 ms; Wilcoxon rank-sum test, two-tailed, Z=−2.86, p=0.004) and longer decay time constant (MDTh, n=8, 54.42±12.78 ms; vHipp, n=5, 17.14±2.72 ms; Wilcoxon rank-sum test, two-tailed, Z=2.56, p=0.010). No difference is detectable in rise time (MDTh, n=8, 3.19±0.51 ms; vHipp, n=5, 3.27±0.79 ms).

Figure 6. Neuropathic pain decreases excitatory and inhibitory currents elicited by MDTh inputs

A, Representative excitatory and inhibitory currents (−70 mV and 0 mV holding potentials, respectively; CS-methanesulfonate + QX-314 intrapipette solution), in response to stimulation of MDTh inputs in sham (green) and SNI (gray). B,C, In SNI slices both excitatory (SNI, n=9, 387±77 pA; sham, n=8, 799±97 pA; Wilcoxon rank-sum test, two-tailed, Z=−2.36, p=0.018) and inhibitory (SNI, n=9, 806±214pA; sham, n=8, 1765±316 pA; Wilcoxon rank-sum test, two-tailed, Z=−2.36, p=0.018) responses are smaller. D, The inhibitory/excitatory current ratio is unaltered in SNI (2.81±0.82, n=9 vs, 2.28±0.35, n=8 in sham; Wilcoxon rank-sum test, two-tailed, Z=0.048, p=0.96). E, Representative currents in response to a pair of blue light stimuli, 100 ms apart, for sham (green) and SNI (gray) conditions (−70 mV holding potential, K-gluconate intrapipette solution). F,G, Paired pulse ratio is not significantly altered in SNI for either a 100 ms inter-pulse interval (SNI, n=15, 1.12±0.08; sham, n=15, 0.97±0.04; Wilcoxon rank-sum test, two-tailed, Z=1.45, p=0.15) or a 200 ms inter-pulse interval (SNI, n=15, 1.00±0.06; sham, n=14, 0.88±0.04; Wilcoxon rank-sum test, two-tailed, Z=1.55, p=0.12).

Figure 7. Neuropathic pain alters the release probability at the vHipp inputs

A, Representative excitatory and inhibitory currents (−70 mV and 0 mV holding potentials, respectively; CS-methanesulfonate + QX-314 intrapipette solution), in response to stimulation of vHipp inputs in sham (purple) and SNI (gray). B,C, The SNI condition does not significantly alter the excitatory (SNI, n=11, 282±58 pA; sham, n=11, 169±43 pA; Wilcoxon rank-sum test, two-tailed, Z=1.38, p=0.17) or inhibitory (SNI, n=10, 429±176 pA; sham, n=9, 127±65 pA; Wilcoxon rank-sum test, two-tailed, Z=1.13, p=0.26) current components. D, The inhibitory/excitatory current ratio is unaltered in SNI (SNI, n=10, 1.47±0.46; sham, n=9, 0.61±0.26; Wilcoxon rank-sum test, two-tailed, Z=1.29, p=0.19). E, Current responses to a pair of blue light stimuli, 100 ms apart, for sham (purple) and SNI (gray) conditions (−70 mV holding potential, K-gluconate intrapipette solution). F,G, Paired pulse ratio becomes significantly facilitating in the SNI condition for both a 100 ms inter-pulse interval (SNI, n=23, 1.33±0.08; sham, n=19 1.00±0.08; Wilcoxon rank-sum test, two-tailed, Z=2.81, p=0.005) and a 200 ms inter-pulse interval (SNI, n=21, 1.39±0.08; sham, n=19, 1.08±0.09; Wilcoxon rank-sum test, two-tailed, Z=2.65, p=0.008).

Figure 8. Proposed organization of vHIPP and MDTh inhibitory inputs to the prelimbic cortex

The schematic summarizes the organization of polysynaptic inhibition in PL layer 5 as suggested by the synaptic delays and kinetic properties of inhibitory currents recorded in pyramidal neurons. Activation of the MDTh input leads to an inhibitory response with large amplitude and short delay (t3) whereas activation of the vHIPP input elicits a response that is smaller in amplitude and with longer delay (t4). Green: MDTh network. Red: vHIPP network.