Description
Cholecystokinin (CCK) was firstly described as a substance within intestinal extracts that can stimulate gallbladder contraction and evacuation (Ivy and Oldberg 1928). Later it was found to be one of the most abundant neuropeptides in the brain (Beinfeld et al. 1981). The bio-active form of CCK, sulfated CCK-8, is thought to regulate varied neuronal processes, including neuronal excitability (Deng et al. 2010; Ma et al. 2006; Wang et al. 2011), anxiety (Daugé and Léna 1998), food intake (Gibbs, Young, and Smith 1973), as well as several types of learning and memory (Huston et al. 1998).Our earlier studies showed that activation of CCK positive neurons that originated from the entorhinal cortex (EC) induces long-term potentiation (LTP) and neuroplasticity in the auditory cortex (Li et al. 2013). Lesions in the EC or the hippocampus will lead to memory deficits including temporal, spatial, episodic memory, as well as associative fear conditioning (Amaral, Zola-Morgan, and Squire 1986) (Squire, Stark, and Clark 2004).
CCK-knock out (CCK-KO) mice show severe deficits in spatial memory and hippocampal LTP formation induced by theta burst stimulation (TBS). In Morris Water Maze Test, comparing to normal behaving control C57 mice, the CCK-KO mice could not shorten the latency used for finding the hidden platform during the 5 days learning period and spent time almost evenly in each quadrant in the probe test on Day 6. TBS-induced LTP in the classical hippocampal CA3-CA1 pathway was also impaired in CCK-KO mice. On the contrary, normal learning curve of platform finding and acquired spatial memory of the hidden platform in CCK Type B Receptor-KO mice group were still observed, along with an intact CA3-CA1 LTP in the acute study. This means CCKBR is not responsible for CCK’s function in hippocampal spatial function and LTP formation. We thus injected CCKAR antagonist Devazepide into hippocampus and found it sufficient to block TBS-LTP in CA3-CA1 pathway, which is consistent with the hippocampus-dense mRNA expression pattern of CCKAR (Datson et al. 2001, Allen Brain Atlas).
CCK positive interneurons are abundant and comprise one of the largest GABAergic neuron groups in hippocampus. Whether these interneurons contribute to TBS-induced hippocampal LTP formation remains unknown. In a recent study, local inhibitory CCK neurons were found to receive input from lateral EC originated long range inhibitory neurons and modulate tri-synaptic LTP in hippocampus (Basu et al. 2016). With AAV-EF1a-DIO-ChR2-EYFP virus injected in hippocampus of CCK-Cre mice, hippocampal CCK positive neurons were manipulated with either low frequency (LF: 1Hz) or high frequency (HF: 80Hz) of 473nm blue light, yet neither LF nor HF blue light stimulation induced LTP in CA3-CA1 pathway. On the contrary, both kinds of manipulation led to strong and mild Long-term Depression (LTD) respectively. We hypothesize the CCK causing hippocampal TBS-LTP is not from local inhibitory neurons but projections of EC, the hub between neocortex and hippocampus. The same virus was then injected to medial and lateral EC, and the CCK positive projections to hippocampus were activated by LF and HF blue light. While LF blue light did not induce LTP, HF blue light, which was paired with LF ES stimulation of CA3 initiated LTP in CA3-CA1 pathway. The nature of these projecting CCK neurons was further studied by immune-histology staining with CamkIIa, an excitatory neuron marker. About 80% of the CCK-positive neurons at the virus injection locus showed co-staining with CamkIIa, indicating a majority of these CCK positive neurons in EC are excitatory. A further study showed HF light activation of EC CCK projection terminals, which expressing CamkIIa-DIO-ChR2 virus, did initiate CA3-CA1 LTP of similar increment to that of expressing EF1a-DIO-ChR2.
At the behavioral level, EC-to-hippocampus CCK projections strengthen the connectivity between place cells after HF blue light stimulation and LF ES pairing. Intra-place cells connection was confirmed in an acute study that ES-TBS successfully caused LTP in longitudinal CA1-CA1 pathway. Then in a spatial fear memory test, three artificial place fields were induced by electrical stimulation through three different electrodes implanted in hippocampus, whenever the mice entered specified regions in a chamber. After days of training, footshock (FS) were given in one of the regions to induce a spatial fear memory of that region, which was represented by the corresponding FS electrode and artificial place field. Under anesthesia, one of the left electrodes was used to pair to the FS electrode with HF blue light stimulation, while the third electrode was paired to FS electrode with LF blue light stimulation or without light. After at least three days of pairing section, the mice showed decreased occupancy in the HF-pairing-corresponding region, while the control region showed no decreased occupancy. This indicates the fear against FS region extended to HF pairing region and HF activation of the EC-to-hippocampus CCK projection enabled intra-place cells association at the behavioral level.
Concluding remarks
In this study, we found that the CCK positive neurons densely project from entorhinal cortex to hippocampus. High frequency stimulation of these CCK terminals/neurons could switch on the long-term potentiation in hippocampal pathways. This LTP was reflected in a spatial fear memory behavioral test. We further demonstrated the activation of CCK-positive neurons in hippocampus does not induce LTP in CA3-CA1 pathway. Overall the study demonstrated a crucial function of EC-to-hippocampus CCK positive projection in the LTP formation in hippocampus.
| Period | 23 Apr 2017 |
|---|---|
| Event title | Minisymposium on Neuropeptides : The diverse roles of cholecystokinin and galanin in the nervous system |
| Event type | Conference |
| Location | GuangzhouShow on map |