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. 2020 Mar 11;40(11):2314-2331.
doi: 10.1523/JNEUROSCI.1919-19.2020. Epub 2020 Jan 31.

Cholecystokinin-Expressing Interneurons of the Medial Prefrontal Cortex Mediate Working Memory Retrieval

Robin Nguyen  1 , Sridevi Venkatesan  2 , Mary Binko  2 , Jee Yoon Bang  3 , Janine D Cajanding  3 , Chloe Briggs  1 , Derya Sargin  2 , Itaru Imayoshi  4 , Evelyn K Lambe  2   5   6 , Jun Chul Kim  7   3
Affiliations

Cholecystokinin-Expressing Interneurons of the Medial Prefrontal Cortex Mediate Working Memory Retrieval

Robin Nguyen et al. J Neurosci. .

Abstract

Distinct components of working memory are coordinated by different classes of inhibitory interneurons in the PFC, but the role of cholecystokinin (CCK)-positive interneurons remains enigmatic. In humans, this major population of interneurons shows histological abnormalities in schizophrenia, an illness in which deficient working memory is a core defining symptom and the best predictor of long-term functional outcome. Yet, CCK interneurons as a molecularly distinct class have proved intractable to examination by typical molecular methods due to widespread expression of CCK in the pyramidal neuron population. Using an intersectional approach in mice of both sexes, we have succeeded in labeling, interrogating, and manipulating CCK interneurons in the mPFC. Here, we describe the anatomical distribution, electrophysiological properties, and postsynaptic connectivity of CCK interneurons, and evaluate their role in cognition. We found that CCK interneurons comprise a larger proportion of the mPFC interneurons compared with parvalbumin interneurons, targeting a wide range of neuronal subtypes with a distinct connectivity pattern. Phase-specific optogenetic inhibition revealed that CCK, but not parvalbumin, interneurons play a critical role in the retrieval of working memory. These findings shine new light on the relationship between cortical CCK interneurons and cognition and offer a new set of tools to investigate interneuron dysfunction and cognitive impairments associated with schizophrenia.SIGNIFICANCE STATEMENT Cholecystokinin-expressing interneurons outnumber other interneuron populations in key brain areas involved in cognition and memory, including the mPFC. However, they have proved intractable to examination as experimental techniques have lacked the necessary selectivity. To the best of our knowledge, the present study is the first to report detailed properties of cortical cholecystokinin interneurons, revealing their anatomical organization, electrophysiological properties, postsynaptic connectivity, and behavioral function in working memory.

Keywords: CCK; GABA; interneuron; mPFC; optogenetics; working memory.

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Figures

Figure 1.
Figure 1.
Intersectional genetic labeling and mPFC distribution of CCK and PV interneurons. A, Dual recombinase-responsive reporter allele, RC::FrePe, containing FRT-flanked and loxP-flanked transcriptional stop cassettes. Middle, FLPe-mediated stop cassette removal results in mCherry expression. Bottom, Additional Cre-mediated excisions remove mCherry and the second stop cassette, resulting in GFP expression. The RC::FrePe allele is knocked in to the Gt(ROSA)26Sor(R26) locus with CAG (chicken β-actin and CMV enhancer) promoter elements. B, Percentage of GFP-labeled cells (i.e., CCK interneurons) that are positive for CCK, GABA, or PV immunoreactivity. N = 3. Data are mean ± SEM. C, Representative confocal images of mPFC from CCK-Dlx5/6-FrePe (left) or PV Dlx5/6-FrePe (right) mice. Green represents GFP in CCK or PV interneurons. Red represents mCherry in CCK-negative or PV-negative GABA interneurons. D, Percentage of the total CCK or PV cells found in each layer. E, Percentage contribution of CCK or PV interneurons to the total GABA interneuron population by layer. N = 3. **p < 0.01, ***p < 0.001. Data are mean ± SEM.
Figure 2.
Figure 2.
Electrophysiological properties of GFP-positive CCK interneurons from CCK-FrePe mice. A, Schematic of the mPFC with the green box representing where whole-cell patch-clamp recordings of GFP-positive CCK interneurons were performed. The IR-DIC image with GFP fluorescence overlay shows an example GFP-positive CCK interneuron. B, Pie chart represents the distribution of GFP-positive CCK non-Fast Spiking (nFS) and Fast-Spiking (FS) interneurons (INs). C, A representative current-clamp trace shows the electrophysiological signature of an nFS IN in response to 25 pA depolarizing and hyperpolarizing current steps. D, A representative current-clamp trace shows the electrophysiological signature of an FS IN in response to 150 pA depolarizing and hyperpolarizing current steps. E, Bar graph represents a more depolarized resting membrane potential (RMP) in nFS INs than FS INs (unpaired t test, t(20) = 3.2, **p = 0.005). F, Bar graph represents a higher input resistance in nFS INs than FS INs (unpaired t test, t(20) = 3.3, **p = 0.004). G, Bar graph shows that the current required to elicit a single action potential is smaller in nFS INs than FS INs (unpaired t test, t(12) = 4.1, **p = 0.002). H, The representative current-clamp traces show the response of an nFS IN and an FS IN to a 200 pA depolarizing step. The representative traces were chosen to illustrate that FS cells need a much higher current step to start firing. I, Graph shows that the percent peak firing is greater in nFS INs (N = 14) than FS INs (N = 5) at lower current injections (two-way ANOVA, effect of cell type, F(1,68) = 18.4, p < 0.0001). **p ≤ 0.01 (Bonferroni's post hoc test). ****p ≤ 0.0001 (Bonferroni's post hoc test). Data are mean ± SEM.
Figure 3.
Figure 3.
Characterization of virally transfected ChR2-eYFP expression in CCK GABAergic interneurons. A, Schematic showing recording site in PFC. Right, Example non-Fast Spiking (nFS) and Fast Spiking (FS) ChR2-eYFP-expressing interneurons in virally transfected mice. The IR-DIC and fluorescence images are shown together for each neuron. B, Pie chart represents the recorded proportions of these two types of CCK interneurons. C, Bar graph shows that FS and nFS interneurons differ significantly in their resting membrane potential (RMP). ***p < 0.001 (unpaired t test). D, Bar graph shows that FS and nFS interneurons also differ significantly in their input resistance. *p < 0.05 (unpaired t test). Example traces showing the sustained action potential firing in a CCK-positive nFS interneuron: (E) to a 100 pA step current injection and (F) to a 10 Hz train of light flashes. Example traces for a CCK-positive FS interneuron showing the responses: (G) to a 300 pA step current injection and (H) to a 10 Hz light train. The representative current-injection traces for FS and nFS interneurons were chosen to illustrate the characteristic high-frequency firing exhibited by FS neurons, which distinguishes them as a group. Data are mean ± SEM.
Figure 4.
Figure 4.
CCK interneurons have widespread inhibitory effects in PFC. A, Illustration of recording site in the mPFC. B, Schematic of experimental paradigm to determine the postsynaptic targets of CCK-positive interneurons by examining light-evoked postsynaptic responses in a variety of neuronal subtypes, including Burst spiking (BS) and Regular Spiking (RS) pyramidal (Pyr), Regular Spiking (RS), Low Threshold (LT) and Fast Spiking (FS) interneurons (Int). C, Histogram represents the latency of the light-evoked IPSCs that were measured in postsynaptic neurons. D, Bar graph represents the amplitude of a subset of these IPSCs before and after the application of the GABA-A receptor antagonist bicuculline (Bic), which significantly reduced the IPSC amplitudes (***p < 0.001, paired t test), confirming that the light-evoked IPSCs are predominantly mediated by GABA-A receptors. A smaller subset of interneurons also had a significant GABA-B component, which was eliminated by the GABA-B antagonist CGP52432 (CGP). Inset, Example IPSC with a significant GABA-B component, which is blocked by combined application of Bic and CGP. Calibration: 50 pA, 100 ms. E, Bar graph represents amplitudes of light-evoked IPSCs measured in different neuronal types. ****p < 0.0001 (one-way ANOVA). F, Bar graph shows how the firing rate of postsynaptic neurons to a depolarizing step was inhibited by the light-evoked excitation of CCK-positive interneurons. ****p < 0.0001 (paired t test). Of note, CCK interneurons could suppress spiking in all their postsynaptic targets. G1–G5, Illustrative action potential firing pattern to step current injection in different types of postsynaptic neurons recorded. H1–H5, Example light-evoked IPSC trace for each type of postsynaptic neuron. Inset, Pie chart represents the connection probability for each group: the proportion of neurons that show light-evoked IPSCs versus no response. I1–I5, Example traces showing the inhibition of spiking in different postsynaptic neurons by CCK interneuron stimulation. The neuronal firing elicited by current injection is shown at baseline (black) and during light activation of CCK-positive interneurons (blue). Data are mean ± SEM.
Figure 5.
Figure 5.
CCK-positive interneurons also release glutamate onto a subset of postsynaptic targets. A, Examples from a postsynaptic interneuron showing an EPSC (blue arrow) that consistently follows the onset of the light-evoked IPSC in successive light flashes. B, Glutamate receptor blockers CNQX and APV selectively inhibit the EPSC, leaving the IPSC intact. C, The further addition of a GABA-A receptor antagonist to the glutamate blockers eliminates the light-evoked postsynaptic responses completely. D, Histogram of latency from light onset demonstrates the respective timing of the light-evoked EPSCs (orange) and IPSCs (blue). The EPSCs occur later than the IPSCs but within 5–9 ms of light onset. E, Pie chart shows the relative proportions of cell types showing light-evoked EPSCs. Regular spiking (RS) interneurons are the most common type that show these light-evoked glutamatergic EPSCs, with examples also seen in a low threshold (LT) interneuron and in regular (RS) and burst spiking (BS) pyramidal (Pyr)neurons.
Figure 6.
Figure 6.
Intersectional genetic expression of ArchT selectively in CCK interneurons. A, Top, Dual recombinase-responsive reporter allele, RC::ArchT, contains two transcriptional stop cassettes flanked by loxP and FRT sites. Bottom, Cre- and FLPe-mediated excisions result in ArchT-EGFP expression in CCK interneurons. B, Representative images of immunofluorescent staining in prelimbic cortex of CCK-ArchT mice for CCK and GABA. C, Representative images of immunofluorescent staining in prelimbic cortex of CCK-ArchT mice for PV and VIP. D, Percentage of ArchT+ cells in the prelimbic cortex (PL) double-labeled with GABA markers. N = 3. Data are mean ± SEM.
Figure 7.
Figure 7.
Electrophysiological properties of GFP-positive CCK interneurons from CCK-ArchT. A, Schematic of the mPFC with the green box representing where whole-cell patch-clamp recordings of GFP-positive CCK interneurons were performed. The IR-DIC image with GFP fluorescence overlay shows an example GFP-positive CCK interneuron. B, The pie chart represents the distribution of GFP-positive CCK non-fast spiking (nFS) and fast-spiking (FS) interneurons (INs). C, A representative voltage-clamp trace at −75 mV shows the outward inhibitory current in response to light from an nFS IN (top) and an FS IN (bottom). Graph shows that the amplitude in response to light is greater in the FS INs compared with the nFS INs (unpaired t test, t(16) = 6.6). ****p < 0.0001. D, A representative current-clamp trace shows the response of an nFS IN to a 175 pA depolarizing step in the absence (black) and presence (blue) of light. The input–output graph shows the firing frequency of nFS INs (N = 8) in response to a series of depolarizing current steps. The firing frequency is reduced in the presence of light (two-way repeated-measures ANOVA, effect of light, F(1,77) = 59.3, p < 0.0001). *p ≤ 0.05 (Bonferroni's post hoc test). **p ≤ 0.01 (Bonferroni's post hoc test). E, A representative current-clamp trace shows the response of an FS IN to a 500 pA depolarizing step in the absence (black) and presence (blue) of light. The input–output graph shows the firing frequency of FS INs (N = 4) in response to a series of depolarizing current steps. The firing frequency is reduced in the presence of light (two-way repeated-measures ANOVA, effect of light, F(1,33) = 59.8, p < 0.0001). ***p ≤ 0.001 (Bonferroni's post hoc test). ****p ≤ 0.0001 (Bonferroni's post hoc test). Data are mean ± SEM.
Figure 8.
Figure 8.
Olfactory DNMS performance. A, Olfactory DNMS apparatus (top view) showing mouse compartment occupation during the sample, delay, and response phases. B, Single-trial test paradigm and response outcomes. C, Percentage of correct responses across increasing delay lengths (N = 6). D, Percentage of false alarms and percentage of misses across increasing delay lengths (N = 6). Data are mean ± SEM.
Figure 9.
Figure 9.
Phase-specific inhibition of mPFC CCK or PV interneurons during DNMS performance. A, Percentage of correct responses across training sessions and number of days to reach criterion performance for CCK-ArchT mice (N = 8) and control mice (N = 10). B, Percentage of correct responses across training sessions and number of days to reach criterion performance for PV-ArchT mice and PV-EYFP mice (N = 6 each). C, Schematic of light illumination over the mPFC at different task phases pseudorandomized across trials with a 5 s delay. D, Representative ArchT expression and diagram of optic fiber placement in prelimbic cortex of CCK-ArchT mice (left), and PV-ArchT mice (right). E–G, CCK-ArchT (N = 8) and control (N = 10) mice performance following light illumination during the sample, delay, and response phases. H–J, PV-ArchT and PV-EYFP mice (N = 6 each) performance following light illumination during the sample, delay, and response phases. E, H, Percentage of correct responses across phases. F, I, Percentage of false alarms across phases. G, J, Percentage of misses across phases. *p < 0.05, ***p < 0.001. Data are mean ± SEM.
Figure 10.
Figure 10.
Phase-specific inhibition of mPFC CCK or PV interneurons during Go/No-go odor discrimination performance. A, Schematic of light illumination at different task phases across trials. B, C, Percentage of correct responses following light illumination during the pseudo-sample, pseudo-delay, and response phases of (B) CCK-ArchT and control (N = 4 each), and (C) PV-ArchT (N = 6) and PV-EYFP (N = 5). Data are mean ± SEM.
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References

    1. Abbas AI, Sundiang MJ, Henoch B, Morton MP, Bolkan SS, Park AJ, Harris AZ, Kellendonk C, Gordon JA (2018) Somatostatin interneurons facilitate hippocampal-prefrontal synchrony and prefrontal spatial encoding. Neuron 100:926–939.e3. 10.1016/j.neuron.2018.09.029 - DOI - PMC - PubMed
    1. Anticevic A, Repovs G, Corlett PR, Barch DM (2011) Negative and nonemotional interference with visual working memory in schizophrenia. Biol Psychiatry 70:1159–1168. 10.1016/j.biopsych.2011.07.010 - DOI - PubMed
    1. Armstrong C, Soltesz I (2012) Basket cell dichotomy in microcircuit function. J Physiol 590:683–694. 10.1113/jphysiol.2011.223669 - DOI - PMC - PubMed
    1. Avdesh A, Hoe Y, Martins RN, Martin-Iverson MT (2013) Pharmacological effects of cannabinoids on the reference and working memory functions in mice. Psychopharmacology (Berl) 225:483–494. 10.1007/s00213-012-2834-6 - DOI - PubMed
    1. Awatramani R, Soriano P, Rodriguez C, Mai JJ, Dymecki SM (2003) Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation. Nat Genet 35:70–75. 10.1038/ng1228 - DOI - PubMed

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