Roles of GABAergic and Glutamatergic Cholecystokinin Neurons in Cortical LTD and LTP
抑制性和興奮性膽囊收縮素神經元在皮層LTD和LTP中的作用
Student thesis: Doctoral Thesis
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Award date | 12 Jan 2021 |
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Permanent Link | https://scholars.cityu.edu.hk/en/theses/theses(5e6dded8-3d47-4f2c-a439-d8f90b4e845e).html |
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Abstract
Synaptic plasticity, which is embodied in the variations of synaptic strength, endows the nervous system with the ability to respond to environmental changes. Two well-known examples of changes in synaptic strength are long-term potentiation (LTP) and long-term depression (LTD), which are now considered to be the neural basis of learning and memory.
Previous studies in our lab demonstrated that neurons in the auditory cortex (AC) began to respond to visual stimuli after repetitive visual-auditory stimuli pairing while inactivation of the entorhinal cortex (EC) impaired this cross-modal associative learning (Chen X et al.,2013). Evidence also showed that the EC contained numerous cholecystokinin-positive (CCK+) neurons, and local infusion of CCK-induced LTP in the AC, while administration of a CCK-B receptor antagonist blocked high-frequency stimulation-induced LTP (Li X et al., 2014). These results indicated an essential role of CCK in learning and memory.
In the present study, we explored how CCK-containing neurons participate in synaptic plasticity. We first demonstrated that CCK depletion impaired associative learning and neuronal plasticity. A brief fear conditioning training was conducted on CCK-knock out (CCK-/-) mice and wild-type (C57) mice to generally assess the roles of CCK in learning and memory. We observed that CCK-/- mice had lower percentages of freezing behavior to conditioned cue when compared with C57 mice. Furthermore, LTD and LTP inductions were absent in CCK-/- mice, indicating a vital function of CCK in both LTD and LTP. With respect to LTD, we first found that low-frequency electrical stimulation (LFES)-induced LTD is mediated by enhanced inhibition. Electrical stimulation (ES)-induced excitatory post-synaptic currents (EPSCs) and inhibitory post-synaptic currents (IPSCs) were recorded from pyramidal neurons using whole-cell patch-clamp recording before and after LFES to measure the alterations of excitatory and inhibitory transmissions following LTD induction. We observed that the amplitude of EPSCs remained stable, while the IPSCs increased dramatically after LFES, indicating an escalation of the inhibitory circuit following LTD induction.
Second, GABAergic CCK neurons contributed to LFES-induced enhancement of inhibition. The source of the enhanced inhibition was examined by measuring inhibitory inputs from two types of perisomatic-targeting interneurons. By combining optogenetics with in vitro whole-cell recording, IPSCs were recorded from pyramidal neurons by laser-activation of Channelrhodopsin (ChR2)-expressing CCK and parvalbumin (PV)-interneurons before and after LFES. The results demonstrated that LFES caused a growth in IPSC amplitudes in both types of interneurons. However, the growth of the amplitudes of IPSCs induced in PV interneurons was blocked when CCK was depleted, indicating that CCK was the key neuromodulator in this inhibitory enhancement.
Third, LFES triggered NR2B subunit-dependent high-frequency firing in GABAergic CCK neurons. CCK interneurons were specifically labeled and patched, and the neuronal activities were recorded before and after LFES to study how GABAergic CCK neurons promoted the strengthening of inhibition. Burst firings (firing spikes at approximately 60 Hz) in GABAergic CCK neurons were observed following LFES. Furthermore, bath application of Ro25-6981 (an NR2B subunit antagonist) and NVP-AAM077 (an NR2A subunit antagonist) indicated that the high-frequency firing of GABAergic CCK neurons was NR2B subunit dependent.
Finally, burst firing of GABAergic CCK neurons led to long-lasting potentiation of inhibition. To explore the effect of high-frequency firing of CCK interneurons on pyramidal neurons, burst firing was mimicked by activating ChR2- expressing CCK interneurons using high-frequency laser stimulation (HFLS). Laser stimulation-induced IPSCs were recorded from pyramidal neurons before and after HFLS, and we found that IPSC amplitudes increased remarkably after HFLS.
With the aspect to LTP, we found that the nature of CCK projections from the EC to the AC was excitatory, and HF laser activation of these CCK-containing terminals enabled LTP in the AC. ChR2 was specifically expressed in CCK+ neurons in the EC, and the responses induced in pyramidal neurons by laser-activation of CCK+ terminals were recorded. We found that laser stimulation of CCK+ terminals induced both EPSCs and IPSCs in the pyramidal neurons of the AC, but the latency of IPSCs was approximately 5 ms longer than that of EPSCs. By delivering tetrodotoxin (TTX) and 4-aminopyridine (4-AP) to isolate monosynaptic input, we found that IPSCs were blocked while EPSCs were preserved, indicating that the IPSCs were derived from the activation of local interneurons and that the monosynaptic CCK projections from the EC to AC were excitatory. Furthermore, HF laser activation of glutamatergic CCK terminals potentiated ES-induced EPSCs in the AC.
In summary, we found that GABAergic and glutamatergic CCK neurons exert indispensable roles in LTD and LTP, respectively. In particular, we demonstrated that LTD, which has been described to be generated by decreased excitability, was mediated by enhanced inhibition following LFES in the cortex. LFES triggered NR2B subunit-dependent burst firing in GABAergic CCK neurons, which supposedly caused CCK release, and therefore generated a prolonged potentiation of inhibition in pyramidal neurons.
Previous studies in our lab demonstrated that neurons in the auditory cortex (AC) began to respond to visual stimuli after repetitive visual-auditory stimuli pairing while inactivation of the entorhinal cortex (EC) impaired this cross-modal associative learning (Chen X et al.,2013). Evidence also showed that the EC contained numerous cholecystokinin-positive (CCK+) neurons, and local infusion of CCK-induced LTP in the AC, while administration of a CCK-B receptor antagonist blocked high-frequency stimulation-induced LTP (Li X et al., 2014). These results indicated an essential role of CCK in learning and memory.
In the present study, we explored how CCK-containing neurons participate in synaptic plasticity. We first demonstrated that CCK depletion impaired associative learning and neuronal plasticity. A brief fear conditioning training was conducted on CCK-knock out (CCK-/-) mice and wild-type (C57) mice to generally assess the roles of CCK in learning and memory. We observed that CCK-/- mice had lower percentages of freezing behavior to conditioned cue when compared with C57 mice. Furthermore, LTD and LTP inductions were absent in CCK-/- mice, indicating a vital function of CCK in both LTD and LTP. With respect to LTD, we first found that low-frequency electrical stimulation (LFES)-induced LTD is mediated by enhanced inhibition. Electrical stimulation (ES)-induced excitatory post-synaptic currents (EPSCs) and inhibitory post-synaptic currents (IPSCs) were recorded from pyramidal neurons using whole-cell patch-clamp recording before and after LFES to measure the alterations of excitatory and inhibitory transmissions following LTD induction. We observed that the amplitude of EPSCs remained stable, while the IPSCs increased dramatically after LFES, indicating an escalation of the inhibitory circuit following LTD induction.
Second, GABAergic CCK neurons contributed to LFES-induced enhancement of inhibition. The source of the enhanced inhibition was examined by measuring inhibitory inputs from two types of perisomatic-targeting interneurons. By combining optogenetics with in vitro whole-cell recording, IPSCs were recorded from pyramidal neurons by laser-activation of Channelrhodopsin (ChR2)-expressing CCK and parvalbumin (PV)-interneurons before and after LFES. The results demonstrated that LFES caused a growth in IPSC amplitudes in both types of interneurons. However, the growth of the amplitudes of IPSCs induced in PV interneurons was blocked when CCK was depleted, indicating that CCK was the key neuromodulator in this inhibitory enhancement.
Third, LFES triggered NR2B subunit-dependent high-frequency firing in GABAergic CCK neurons. CCK interneurons were specifically labeled and patched, and the neuronal activities were recorded before and after LFES to study how GABAergic CCK neurons promoted the strengthening of inhibition. Burst firings (firing spikes at approximately 60 Hz) in GABAergic CCK neurons were observed following LFES. Furthermore, bath application of Ro25-6981 (an NR2B subunit antagonist) and NVP-AAM077 (an NR2A subunit antagonist) indicated that the high-frequency firing of GABAergic CCK neurons was NR2B subunit dependent.
Finally, burst firing of GABAergic CCK neurons led to long-lasting potentiation of inhibition. To explore the effect of high-frequency firing of CCK interneurons on pyramidal neurons, burst firing was mimicked by activating ChR2- expressing CCK interneurons using high-frequency laser stimulation (HFLS). Laser stimulation-induced IPSCs were recorded from pyramidal neurons before and after HFLS, and we found that IPSC amplitudes increased remarkably after HFLS.
With the aspect to LTP, we found that the nature of CCK projections from the EC to the AC was excitatory, and HF laser activation of these CCK-containing terminals enabled LTP in the AC. ChR2 was specifically expressed in CCK+ neurons in the EC, and the responses induced in pyramidal neurons by laser-activation of CCK+ terminals were recorded. We found that laser stimulation of CCK+ terminals induced both EPSCs and IPSCs in the pyramidal neurons of the AC, but the latency of IPSCs was approximately 5 ms longer than that of EPSCs. By delivering tetrodotoxin (TTX) and 4-aminopyridine (4-AP) to isolate monosynaptic input, we found that IPSCs were blocked while EPSCs were preserved, indicating that the IPSCs were derived from the activation of local interneurons and that the monosynaptic CCK projections from the EC to AC were excitatory. Furthermore, HF laser activation of glutamatergic CCK terminals potentiated ES-induced EPSCs in the AC.
In summary, we found that GABAergic and glutamatergic CCK neurons exert indispensable roles in LTD and LTP, respectively. In particular, we demonstrated that LTD, which has been described to be generated by decreased excitability, was mediated by enhanced inhibition following LFES in the cortex. LFES triggered NR2B subunit-dependent burst firing in GABAergic CCK neurons, which supposedly caused CCK release, and therefore generated a prolonged potentiation of inhibition in pyramidal neurons.
- cholecystokinin, auditory cortex, entorhinal cortex, long-term depression, long-term potentiation