Studying The Effect Of GHS-R1a On Hippocampus-dependent Memory Formation By Selectively Manipulating Its Expression In Excitatory/inhibitory Neurons In Mice

Growth hormone secretagogue receptor 1a(GHS-R1a)is a recognized receptor for nutrition-sensing gastrointestinal peptide hormone ghrelin.GHS-R1a is distributed in the hypothalamus and other regions close related to memory and emotion,such as the hippocampus,the prefrontal cortex,the amygdala,etc.Therefore,GHS-R1a may be involved in the regulation of learning and memory,anxiety and depression,and associated with the pathophysiology of multiple cognitive and affective disorders.In addition to being activated by ghrelin,GHS-R1a has a high,ligand-independent autonomous activity.Although previous studies have reported that ghrelin regulates learning and memory,the conclusions are currently contradictory.So far,the precise mechanisms of ghrelin and GHS-R1a regulating learning and memory are still uncertain.Under normal physiological conditions,only trace amount of endogenous ghrelin can be detected in the central nervous system,thus the autonomous activity of GHS-R1a may be one of the important ways to regulate neuronal function.In previous studies,we found that Ghsr1a knockout(Ghsr1a KO)reduced the excitability of GAD67~+inhibitory neurons in the dCA1 region of the hippocampus,meanwhile the mutant mice exhibited hippocampus-dependent memory enhancement.In addition,overexpression of GHS-R1a inαCaMKII~+neurons in hippocampal dCA1 region inhibited excitability of CA1 pyramidal neurons,and the overexpressed mice showed spatial and positional memory impairment.Therefore,we speculated that GHS-R1a signaling differentially regulates the activity of excitability and inhibitory neurons in the hippocampus.This neuronal type-specific regulation may be critical for GHS-R1a to regulate memory.In order to explore the effect of GHS-R1a on learning and memory,we first injected pAAV-hSyn-DIO-Ghsr1a-EGFP virus or control virus into Camk2a-Cre、Vglut1-Cre and Dlx5/6-Cre mice respectively.The effect of selective overexpress of GHS-R1a in hippocampal excitatory/inhibitory neurons on hippocampus-dependent learning and memory was then investigated.The behavioral paradigms used in our study included elevated plus-maze(EPM)test,open field(OF)test,new object recognition(NOR),new object-place recognition(NPR),and Morris water maze(MWM).Moreover,we injected chemogenetic virus pAAV-Camk2a-HA-hM4D(Gi)-2A-Ghsr1a-sfGFP or control virus Camk2a-HA-h M4D(Gi)-2A-sfGFP into the hippocampal dCA1 area.Virus-infected dCA1αCaMKII~+neurons thus could be temporarily inactivated by i.p.injection of clozapine-N-oxide(CNO).Using this chemogenetic tool,we further investigated the effect of inactivating dCA1αCaMKII~+neurons with or without GHS-R1a overexpression on memory retrieval.Our main findings are listed as follows:1.Overexpression of GHS-R1a inαCaMKII~+or VGLUT1~+excitatory neurons in the dCA1 region of the hippocampus impairs hippocampus-dependent learning and memory in Camk2a-Cre or Vglut1-Cre mice respectively.1.1.A NOR memory test showed that virus-mediated Ghsr1a overexpression inαCaMKII~+or VGLUT1~+neuron in the hippocampal dCA1 impaired object recognition memory.Specifically,Camk2a-Cre mice with GHS-R1a overexpression showed significantly less time exploring the new object than that the control Camk2a-Cre mice.Consistently,Vglut1-Cre mice with Ghsr1a overexpression spent similar time exploring new and old objects,indicating object recognition memory impairments.Ghsr1a-overexpressed mice and control mice showed similar total object-exploration time,indicating that GHS-R1a overexpression did not affect the exploration activities in mice.1.2.A NPR memory test showed that virus-mediated Ghsr1a overexpression inαCaMKII~+neuron in the hippocampal dCA1 region impaired object-place recognition memory.Specifically,Camk2a-Cre mice with GHS-R1a overexpression spent similar time exploring object in new place and object in old place,indicating object-place recognition memory impairment.Ghsr1a-overexpressed mice and control mice showed similar total object-place exploration time.1.3.A MWM test showed that virus-mediated Ghsr1a overexpression inαCaMKII~+or VGLUT1~+neurons in the hippocampal dCA1 region impaired spatial memory.Specifically,both Camk2a-Cre mice with Ghsr1a overexpression and Vglut1-Cre mice with Ghsr1a overexpression exhibited similar percentage of exploration time in the target quadrant as in other quadrants,while control mice exhibited higer percentage of exploration time in the target quadrant than in other quadrants.There was no significant difference in the latency time to platform and swimming speed among Ghsr1a-overexpressed mice and control mice,indicating that GHS-R1a overexpression did not affect the spatial learning and motor coordination.1.4.An EPM test and An OF test showed that virus-mediated Ghsr1a overexpression inαCaMKII~+or VGLUT1~+excitatory neurons in the dCA1 region of the hippocampus does not affect basal anxiety and locomotor activity of the mice.Specifically,Ghsr1a-overexpressed mice and control mice displayed similar time exploring the open and closed arms in an EPM test,and similar time exploring the center and peripheral area in an OF test.Therefore,the suppressive effect of GHS-R1a overexpression on hippocampus-dependent memory formation was not due to changes in spontaneous activity or stress response.2.Overexpression of DLX5/6~+inhibitory neurons GHS-R1a in the dCA1 region of the hippocampus promotes hippocampus-dependent learning and memory in mice.2.1.A NPR test showed that Ghsr1a overexpression in hippocampal dCA1 DLX5/6~+inhibitory neurons promoted object-place recognition memory in mice.Specifically,Ghsr1a-overexpressed mice spent more percentage of time exploring objects in new place than object in old place,while control mice exhibited similar time exploring both objects with the same training conditions.Two groups of mice displayed similar total object-exploration time.2.2.A MWM test showed that Ghsr1a overexpression in hippocampal dCA1 DLX5/6~+inhibitory neurons improved spatial memory.At day 7 probe test,Ghsr1a-overexpressed mice exhibited more percentage of exploration time in the target quadrant than in other quadrants,while under the control mice did not with the same training condition.There was no significant difference in latency time to platform and swimming speed between the two groups of mice.2.3.An EPM test and an OF test showed that Ghsr1a overexpression in hippocampal dCA1 DLX5/6~+inhibitory neurons did not affect basal anxiety or spontaneous activity of mice.Thus the promoting effect of GHS-R1a overexpression on hippocampus-dependent memory formation was not due to changes in spontaneous activity or stress response.3.Effect of chemogenetic inactivation of hippocampal dCA1αCaMKII~+neurons with or without GHS-R1a overexpression on memory retrieval.3.1.A NOR memory test showed that Ghsr1a overexpression in dCA1αCaMKII~+neurons impaired object recognition memory,which could be improved by intensive training.Intraperitoneal injection of CNO before test blocked the retrival of object recognition memory in control mice,while had no effect on Ghsr1a–overexpressed mice.Specifically,control mice showed similar time exploring the two objects after CNO administration,while Ghsr1a–overexpressed mice still prefered new object than old object.3.2.A NPR test showed that Ghsr1a overexpression in dCA1αCaMKII~+neurons impaired object-place recognition memory,which could be improved by intensive training.Intraperitoneal injection of CNO before test blocked the retrival of object-place memory in control mice,while had no effect on Ghsr1a–overexpressed mice.Specifically,control mice showed similar time exploring the two objects after CNO administration,while Ghsr1a–overexpressed mice still prefered the object in new place than that in old place.3.3.A MWM test showed that Ghsr1a overexpression in dCA1αCaMKII~+neurons impaired spatial memory,which could be improved by intensive training.Intraperitoneal injection of CNO before probe test blocked the retrival of spatial memory in control mice,while had no effect on Ghsr1a–overexpressed mice.Specifically,control mice showed similar percentage exploration time in the target quadrant as in other quadrants after CNO administration,while Ghsr1a–overexpressed mice still spent significantly more time exploring the target quadrant than other quadrants.In summary,we found that Ghsr1a overexpression inαCaMKII~+or VGLUT1~+excitatory neurons in the dCA1 region of the hippocampus inhibited,while Ghsr1a overexpression in dCA1 DLX5/6~+inhibitory neurons enhanced hippocampus-dependent memory,indicating that GHS-R1a signaling in dCA1 excitatory pyramidal neurons and inhibitory interneurons plays opposite role in regulating learning and memory.Intensive training can eliminate the inhibitory effect of selective GHS-R1a overexpression inαCaMKII~+neurons on hippocampal-dependent memory.Chemogenetic inactivation ofαCaMKII~+neurons with GHS-R1a overexpression do not affect memory retrieval,indicating that the memory is not stored in this subpopulation of neurons,that is to say,this population of neurons overexpressing GHS-R1a may not participate in memory encoding.In contrast,we found that chemogenetic inactivation ofαCaMKII~+neurons in the dCA1region in control mice(without GHS-R1a overexpression)blocked memory retrival,indicating that this subpopulation ofαCaMKII~+neurons without GHS-R1a overexpression participates memory encoding,that is to say,this population ofαCaMKII~+neurons is engram cells in the hippocampal dCA1 region.Therefore,our results suggest that GHS-R1a overexpression in someαCaMKII~+excitatory neurons in the hippocampal dCA1region restricts those neurons to participate in memory encoding GHS-R1a overexpression makes neighboring neurons with less GHS-R1a expression,or even neurons outside the dCA1 regions more easily to participate in memory coding,so inactivating those GHS-R1a-overexpressedαCa MKII~+neurons by CNO injection does not affect retrieval of a specific memory.Through the above research,we believe that GHS-R1a can achieve precise regulation of hippocampal memory neural circuit activity and memory-related behaviors by acting on different types of hippocampal neurons(excitability/inhibitory).The regulation of GHS-R1a on hippocampal-dependent memory coding is close related to its regulation on neuronal excitability.These innovative discoveries not only deepen our understanding of the multiple physiological functions and mechanisms of ghrelin and GHS-R1a,but also provide a theoretical basis for the development of drugs that target GHS-R1a to improve memory.