Long-Range Connections Of GABAergic Neurons In The Mice Zona Incerta

The zona incerta(ZI)is a nucleus in the lateral hypothalamus composed mainly of GABAergic neurons with extensive connections throughout the brain.It can affect the activity pattern of cortical neurons and bidirectional control of behavioral response,and may serve as an integrative node of the circuits for global behavioral modulation.It is also involved in various functions such as binge-eating,predatory hunting,sleeping,investigatory and seeking behavior,fear memory,and neuropathic pain.ZI is typically divided into four sectors,and the inputs,outputs,and functions of different sectors vary greatly.At present,the research on ZI input-output circuits is still insufficient,and there is a lack of comparison of different sectors and analysis of projection patterns at the single neuron level,which hinder the understanding of ZI functional mechanism.A series of studies have been carried out in this study to address the above shortcomings.(1)The input maps of different types and sectors of neurons in the ZI were obtained.Parvalbumin,nitric oxide synthase 1,and GABAergic neurons had similar inputs.The input data sets of GABAergic neurons in the rostral(ZIr),intermediomedial(ZIim),intermediolateral(ZIil),and caudal(ZIc)sectors of ZI were obtained by virus tools and fluorescence micro-optical sectioning tomography(f MOST).ZI received input primarily from the isocortex,hypothalamus,and midbrain,and to a lesser extent from the striatum,pallidus,thalamus,pons,medulla,and cerebellar nuclei.The input of four ZI sectors varies greatly,and cortical input regions could be divided into three categories: anterior limbic cortex,motor cortex,and sensory cortex.ZIr and ZIim had similar inputs,both preferring input from the frontal cortex and hypothalamus.ZIil and ZIc had similar inputs,both preferring input from the sensory cortex and midbrain.(2)The output maps of GABAergic neurons in four sectors of ZI were obtained,the organization patterns of outputs were explored,and the preferences of input and output were described.Virus tools and f MOST were used to obtain output data sets of four sectors of ZI,and it was found that ZI output mainly to thalamus,hypothalamus and midbrain,and a small amount to the pons and medulla.ZIr and ZIim had similar outputs,both preferring output to the thalamus and hypothalamus.ZIil and ZIc had similar outputs,both preferring output to the thalamus and midbrain.The outputs of four ZI sectors were quite different,and sometimes they were distributed in different subregions of a same brain region.The efferent fibers of four ZI sectors were distributed in the periaqueductal gray(PAG)and the superior colliculus(SC)in a fan shape with the aqueduct as the center.Using retrograde tracers,neuronal cell bodies projecting to the ventral PAG and ventrolateral SC were found to be distributed in different subregions of the ZIim,and the outputs of these two types of neurons were quite different.By comparing the input and output,it was found that the input of ZI was more extensive,and the output was more concentrated.ZI prefered input from the cerebrum and output to the thalamus.There was also a preference for connections between different sectors of ZI.The ZIr received projections from the dorsal ZIil and almost no projections from the ZIim and ventral ZIil.(3)The projection patterns at the single neuron level were analyzed,and it was found that neuronal cell bodies with similar projection patterns tended to be distributed together.Using virus tool and f MOST,43 long-range projection neurons of ZI were labeled and reconstructed,and it was found that the neurons in ZI had rich and complex efferent fibers.Some neurons projected to dozens of target regions simultaneously through complex pathways,while others projected to only a few brain regions.Combining the results of output and neuron morphology,it suggested that ZI may contain many small subregions with different outputs.In conclusion,the input,output and single neuron projection patterns of ZI GABAergic neurons were analyzed by using a variety of neural circuit tracing tools,and the whole brain connection rules were summarized.The whole-brain connectivity map lays a solid foundation for further studies on the function of ZI.Whole-brain connectivity patterns and single-neuron-level projection patterns provide new insights into its global roles.