Severely Impaired Learning and Altered Neuronal Morphology in Mice Lacking NMDA Receptors in Medium Spiny Neurons

The striatum is composed predominantly of medium spiny neurons (MSNs) that integrate excitatory, glutamatergic inputs from the cortex and thalamus, and modulatory dopaminergic inputs from the ventral midbrain to influence behavior. Glutamatergic activation of AMPA, NMDA, and metabotropic receptors on MSNs is important for striatal development and function, but the roles of each of these receptor classes remain incompletely understood. Signaling through NMDA-type glutamate receptors (NMDARs) in the striatum has been implicated in various motor and appetitive learning paradigms. In addition, signaling through NMDARs influences neuronal morphology, which could underlie their role in mediating learned behaviors. To study the role of NMDARs on MSNs in learning and in morphological development, we generated mice lacking the essential NR1 subunit, encoded by the Grin1 gene, selectively in MSNs. Although these knockout mice appear normal and display normal 24-hour locomotion, they have severe deficits in motor learning, operant conditioning and active avoidance. In addition, the MSNs from these knockout mice have smaller cell bodies and decreased dendritic length compared to littermate controls. We conclude that NMDAR signaling in MSNs is critical for normal MSN morphology and many forms of learning

Knockout mice fail to learn a simple (FR1) operant task.

<p>(<i>A</i>) Lever presses during operant conditioning sessions by control (n = 10) and knockout (n = 5) animals across days; two-way, repeated-measures ANOVA: genotype effect <i>F</i>(1, 13) = 15.72, <i>P</i> = 0.002; day effect <i>F</i>(6, 78) = 10.90, <i>P</i><0.001; genotype x day effect <i>F</i>(6,78) = 12.96, <i>P</i><0.001 (###<i>P</i><0.001); **<i>P</i><0.01, ***<i>P</i><0.001 compared to session 1 within genotype. (<i>B</i>) Latency to first lever press during operant conditioning sessions by control (n = 10) and knockout (n = 5) animals across days; two-way repeated measures ANOVA: genotype effect <i>F</i>(1, 13) = 38.75, <i>P</i><0.001; day effect <i>F</i>(6, 78) = 2.26, <i>P</i> = 0.04; genotype x day effect <i>F</i>(6,78) = 8.04, <i>P</i><0.001 (###<i>P</i><0.001); **<i>P</i><0.01, ***<i>P</i><0.001 compared to session 1 within genotype. (<i>C</i>) Total number of head entries during operant conditioning sessions by control (n = 10) and knockout (n = 5) animals across days; two-way repeated measures ANOVA: genotype effect <i>F</i>(1, 13) = 10.14, <i>P</i> = 0.007; day effect <i>F</i>(6, 78) = 1.93, <i>P</i> = 0.08; genotype x day effect <i>F</i>(6,78) = 2.25, <i>P</i> = 0.04 (#<i>P</i><0.05); *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 compared to session 1 within genotype.</p

Knockout mice that selectively lack NMDAR in striatal MSNs have abnormal MSN morphology.

<p><i>(A</i>) GPR88-CreGFP is expressed selectively in the striatum of knockout animals. A DAPI counterstain was performed. (<i>B</i>) NR1 Western blot of striatal (str) and cortical (cx) homogenates from control and knockout animals. (<i>C)</i> Representative micrographs and tracings of MSNs from control and knockout animals (20 µm scale bar). (<i>D)</i> Cell body size of MSNs in control and knockout mice (n = 18 neurons per genotype); <i>P</i> = 0.009 by unpaired t test; **<i>P</i><0.01. (<i>E)</i> Total MSN dendrite length in control and knockout mice (n = 18 neurons per genotype); <i>P</i> = 0.002 by unpaired t test; **<i>P</i><0.01. (<i>F)</i> Number of dendrites by dendrite order in control and knockout mice (n = 18 neurons per genotype); *<i>P</i><0.05 by unpaired t test. Diagram in (<i>F</i>) depicts the definition of dendrite order.</p

Knockout mice exhibit impaired locomotor learning.

<p>(<i>A</i>). Rotarod performance by control (n = 12) and knockout (n = 11) animals across days; two-way, repeated-measures ANOVA: genotype effect <i>F</i>(1, 21) = 70.5, <i>P</i><0.001; day effect <i>F</i>(8, 168) = 1.63, <i>P</i> = 0.12; genotype x day effect <i>F</i>(8, 168) = 2.37, <i>P</i> = 0.02 (#<i>P</i><0.05); *<i>P</i><0.05, **<i>P</i><0.01, **<i>P</i><0.001 compared to trial 1 within genotype. (<i>B</i>) Latency to fall during a wire-grip test in control (n = 12) and knockout (n = 11) animals; <i>P</i> = 0.08 by unpaired t test.</p

Knockout mice are smaller than normal but have normal spontaneous locomotion, and intact exploratory behavior.

<p><i>(A)</i> Spontaneous locomotion by control (n = 10) and knockout (n = 11) animals; light cycle, <i>P</i> = 0.85; dark cycle, <i>P</i> = 0.93; total, <i>P</i> = 0.98 by unpaired t tests. (<i>B</i>). Body weight of 8-week-old animals; control (n = 15) and knockout (n = 12) males, <i>P</i><0.0001; control (n = 11) and knockout (n = 10) females, <i>P</i> = 0.002 by unpaired t tests; **<i>P</i><0.01; ***<i>P</i><0.0001. (<i>C</i>) Locomotor activity of control (n = 10) and knockout (n = 5) animals in an open field; <i>P</i> = 0.17 by unpaired t test. (<i>D</i>) Fraction of time spent in the center and periphery of the open field by control (n = 10) and knockout (n = 5) animals; <i>P</i> = 0.08 by unpaired t tests. (<i>E</i>) Number of open-field center crossings by control (n = 10) and knockout (n = 5) animals; <i>P</i> = 0.28 by unpaired t test.</p

Cross-species comparative analysis of single presynapses

Abstract Comparing brain structure across species and regions enables key functional insights. Leveraging publicly available data from a novel mass cytometry-based method, synaptometry by time of flight (SynTOF), we applied an unsupervised machine learning approach to conduct a comparative study of presynapse molecular abundance across three species and three brain regions. We used neural networks and their attractive properties to model complex relationships among high dimensional data to develop a unified, unsupervised framework for comparing the profile of more than 4.5 million single presynapses among normal human, macaque, and mouse samples. An extensive validation showed the feasibility of performing cross-species comparison using SynTOF profiling. Integrative analysis of the abundance of 20 presynaptic proteins revealed near-complete separation between primates and mice involving synaptic pruning, cellular energy, lipid metabolism, and neurotransmission. In addition, our analysis revealed a strong overlap between the presynaptic composition of human and macaque in the cerebral cortex and neostriatum. Our unique approach illuminates species- and region-specific variation in presynapse molecular composition