Yunping Deng

Corticostriatal projection neurons - dichotomous types and dichotomous functions

The mammalian striatum receives its main excitatory input from the two types of cortical pyramidal neurons of layer 5 of the cerebral cortex – those with only intratelencephalic connections (IT-type) and those sending their main axon to the brainstem via the pyramidal tract (PT-type). These two neurons types are present in layer 5 of all cortical regions, and thus they appear to project together to all parts of striatum. These two neuron types, however, differ genetically, morphologically, and functionally, with IT-type neurons conveying sensory and motor planning information to striatum and PT-type neurons conveying an efference copy of motor commands (for motor cortex at least). Anatomical and physiological data for rats, and more recent data for primates, indicate that these two cortical neuron types also differ in their targeting of the two main types of striatal projection neurons, with the IT-type input preferentially innervating direct pathway neurons and the PT-type input preferentially innervating indirect pathway striatal neurons. These findings have implications for understanding how the direct and indirect pathways carry out their respective roles in movement facilitation and movement suppression, and they have implications for understanding the role of corticostriatal synaptic plasticity in adaptive motor control by the basal ganglia.

CAG repeat lengths ≥335 attenuate the phenotype in the R6/2 Huntington’s disease transgenic mouse

With spontaneous elongation of the CAG repeat in the R6/2 transgene to > or =335, resulting in a transgene protein too large for passive entry into nuclei via the nuclear pore, we observed an abrupt increase in lifespan to >20 weeks, compared to the 12 weeks common in R6/2 mice with 150 repeats. In the > or =335 CAG mice, large ubiquitinated aggregates of mutant protein were common in neuronal dendrites and perikaryal cytoplasm, but intranuclear aggregates were small and infrequent. Message and protein for the > or =335 CAG transgene were reduced to one-third that in 150 CAG R6/2 mice. Neurological and neurochemical abnormalities were delayed in onset and less severe than in 150 CAG R6/2 mice. These findings suggest that polyQ length and pathogenicity in Huntingtons disease may not be linearly related, and pathogenicity may be less severe with extreme repeats. Both diminished mutant protein and reduced nuclear entry may contribute to phenotype attenuation.

A Novel Closed-Head Model of Mild Traumatic Brain Injury Caused by Primary Overpressure Blast to the Cranium Produces Sustained Emotional Deficits in Mice

Emotional disorders are a common outcome from mild traumatic brain injury (TBI) in humans, but their pathophysiological basis is poorly understood. We have developed a mouse model of closed-head blast injury using an air pressure wave delivered to a small area on one side of the cranium, to create mild TBI. We found that 20-psi blasts in 3-month-old C57BL/6 male mice yielded no obvious behavioral or histological evidence of brain injury, while 25–40 psi blasts produced transient anxiety in an open field arena but little histological evidence of brain damage. By contrast, 50–60 psi blasts resulted in anxiety-like behavior in an open field arena that became more evident with time after blast. In additional behavioral tests conducted 2–8 weeks after blast, 50–60 psi mice also demonstrated increased acoustic startle, perseverance of learned fear, and enhanced contextual fear, as well as depression-like behavior and diminished prepulse inhibition. We found no evident cerebral pathology, but did observe scattered axonal degeneration in brain sections from 50 to 60 psi mice 3–8 weeks after blast. Thus, the TBI caused by single 50–60 psi blasts in mice exhibits the minimal neuronal loss coupled to “diffuse” axonal injury characteristic of human mild TBI. A reduction in the abundance of a subpopulation of excitatory projection neurons in basolateral amygdala enriched in Thy1 was, however, observed. The reported link of this neuronal population to fear suppression suggests their damage by mild TBI may contribute to the heightened anxiety and fearfulness observed after blast in our mice. Our overpressure air blast model of concussion in mice will enable further studies of the mechanisms underlying the diverse emotional deficits seen after mild TBI.

Loss of Corticostriatal and Thalamostriatal Synaptic Terminals Precedes Striatal Projection Neuron Pathology in Heterozygous Q140 Huntington’s Disease Mice

Motor slowing, forebrain white matter loss, and striatal shrinkage have been reported in premanifest Huntingtons disease (HD) prior to overt striatal neuron loss. We carried out detailed LM and EM studies in a genetically precise HD mimic, heterozygous Q140 HD knock-in mice, to examine the possibility that loss of corticostriatal and thalamostriatal terminals prior to striatal neuron loss underlies these premanifest HD abnormalities. In our studies, we used VGLUT1 and VGLUT2 immunolabeling to detect corticostriatal and thalamostriatal (respectively) terminals in dorsolateral (motor) striatum over the first year of life, prior to striatal projection neuron pathology. VGLUT1+ axospinous corticostriatal terminals represented about 55% of all excitatory terminals in striatum, and VGLUT2+ axospinous thalamostriatal terminals represented about 35%, with VGLUT1+ and VGLUT2+ axodendritic terminals accounting for the remainder. In Q140 mice, a significant 40% shortfall in VGLUT2+ axodendritic thalamostriatal terminals and a 20% shortfall in axospinous thalamostriatal terminals were already observed at 1 month of age, but VGLUT1+ terminals were normal in abundance. The 20% deficiency in VGLUT2+ thalamostriatal axospinous terminals persisted at 4 and 12 months in Q140 mice, and an additional 30% loss of VGLUT1+ corticostriatal terminals was observed at 12 months. The early and persistent deficiency in thalamostriatal axospinous terminals in Q140 mice may reflect a development defect, and the impoverishment of this excitatory drive to striatum may help explain early motor defects in Q140 mice and in premanifest HD. The loss of corticostriatal terminals at 1 year in Q140 mice is consistent with prior evidence from other mouse models of corticostriatal disconnection early during progression, and can explain both the measurable bradykinesia and striatal white matter loss in late premanifest HD.

Differential localization of the GluR1 and GluR2 subunits of the AMPA-type glutamate receptor among striatal neuron types in rats.

Differences among the various striatal projection neuron and interneuron types in cortical input, function, and vulnerability to degenerative insults may be related to differences among them in AMPA-type glutamate receptor abundance and subunit configuration. We therefore used immunolabeling to assess the frequency and abundance of GluR1 and GluR2, the most common AMPA subunits in striatum, in the main striatal neuron types. All neurons projecting to the external pallidum (GPe), internal pallidum (GPi) or substantia nigra, as identified by retrograde labeling, possessed perikaryal GluR2, while GluR1 was more common in striato-GPe than striato-GPi perikarya. The frequency and intensity of immunostaining indicated the rank order of their perikaryal GluR1:GluR2 ratio to be striato-GPe>striatonigral>striato-GPi. Ultrastructural studies suggested a differential localization of GluR1 and GluR2 to striatal projection neuron dendritic spines as well, with GluR1 seemingly more common in striato-GPe spines and GluR2 more common in striato-GPi and/or striatonigral spines. Comparisons among projection neurons and interneurons revealed GluR1 to be most common and abundant in parvalbuminergic interneurons, and GluR2 most common and abundant in projection neurons, with the rank order for the GluR1:GluR2 ratio being parvalbuminergic interneurons>calretinergic interneurons>cholinergic interneurons>projection neurons>somatostatinergic interneurons. Striosomal projection neurons had a higher GluR1:GluR2 ratio than did matrix projection neurons. The abundance of both GluR1 and GluR2 in striatal parvalbuminergic interneurons and projection neurons is consistent with their prominent cortical input and susceptibility to excitotoxic insult, while differences in GluR1:GluR2 ratio among projection neurons are likely to yield differences in Ca(2+) permeability, desensitization, and single channel current, which may contribute to differences among them in plasticity, synaptic integration, and excitotoxic vulnerability. The apparent association of the GluR1 subunit with synaptic plasticity, in particular, suggests striato-GPe neuron spines as a particular site of corticostriatal synaptic plasticity, presumably associated with motor learning.

Striatal parvalbuminergic neurons are lost in Huntington's disease: implications for dystonia

Although dystonia represents a major source of motor disability in Huntingtons disease (HD), its pathophysiology remains unknown. Because recent animal studies indicate that loss of parvalbuminergic (PARV+) striatal interneurons can cause dystonia, we investigated if loss of PARV+ striatal interneurons occurs during human HD progression, and thus might contribute to dystonia in HD. We used immunolabeling to detect PARV+ interneurons in fixed sections, and corrected for disease‐related striatal atrophy by expressing PARV+ interneuron counts in ratio to interneurons co‐containing somatostatin and neuropeptide Y (whose numbers are unaffected in HD). At all symptomatic HD grades, PARV+ interneurons were reduced to less than 26% of normal abundance in rostral caudate. In putamen rostral to the level of globus pallidus, loss of PARV+ interneurons was more gradual, not dropping off to less than 20% of control until grade 2. Loss of PARV+ interneurons was even more gradual in motor putamen at globus pallidus levels, with no loss at grade 1, and steady grade‐wise decline thereafter. A large decrease in striatal PARV+ interneurons, thus, occurs in HD with advancing disease grade, with regional variation in the loss per grade. Given the findings of animal studies and the grade‐wise loss of PARV+ striatal interneurons in motor striatum in parallel with the grade‐wise appearance and worsening of dystonia, our results raise the possibility that loss of PARV+ striatal interneurons is a contributor to dystonia in HD.

Single-cell RT-PCR, in situ hybridization histochemical, and immunohistochemical studies of substance P and enkephalin co-occurrence in striatal projection neurons in rats.

Single-cell RT-PCR studies in 3-4-week-old rats have raised the possibility that as many as 20% of striatal projection neurons may be a unique type that contains both substance P (SP) and enkephalin (ENK). We used single-cell RT-PCR, retrograde labeling, in situ hybridization histochemistry, and immunolabeling to characterize the abundance of this cell type, its projection target(s), and any developmental changes in its frequency. We found by RT-PCR that 11% of neurons containing either SP or ENK contained both in 4-week-old rats, while in 4-month-old rats SP/ENK colocalization was only 3%. SP-only neurons tended to co-contain dynorphin and ENK-only neurons neurotensin, while SP/ENK neurons tended to contain dynorphin. Single-cell RT-PCR showed SP/ENK co-occurrence in 4-week-old rats to be no more common among striatal neurons retrogradely labeled from the substantia nigra than among those retrogradely labeled from globus pallidus. Double-label in situ hybridization showed SP/ENK perikarya to be scattered throughout striatum, making up 8% of neurons containing either SP or ENK at 4 weeks, but only 4% at 4 months. Immunolabeling showed that presumptive striatal terminals in globus pallidus externus, globus pallidus internus and substantia nigra pars reticulata that colocalized SP and ENK were scarce. Terminals colocalizing SP and ENK were, however, abundant in the substantia nigra pars compacta. Thus, SP-only and ENK-only neurons make up the vast majority of striatal projection neurons in rats, the frequency of SP/ENK colocalizing striatal neurons is low in adult rats (3-4%), and SP/ENK colocalizing neurons primarily project to SNc but do not appear to be confined to striosomes.

The group 2 metabotropic glutamate receptor agonist LY379268 rescues neuronal, neurochemical and motor abnormalities in R6/2 Huntington's disease mice

Excitotoxic injury to striatum by dysfunctional cortical input or aberrant glutamate uptake may contribute to Huntingtons disease (HD) pathogenesis. Since corticostriatal terminals possess mGluR2/3 autoreceptors, whose activation dampens glutamate release, we tested the ability of the mGluR2/3 agonist LY379268 to improve the phenotype in R6/2 HD mice with 120-125 CAG repeats. Daily subcutaneous injection of a maximum tolerated dose (MTD) of LY379268 (20mg/kg) had no evident adverse effects in WT mice, and diverse benefits in R6/2 mice, both in a cohort of mice tested behaviorally until the end of R6/2 lifespan and in a cohort sacrificed at 10weeks of age for blinded histological analysis. MTD LY379268 yielded a significant 11% increase in R6/2 survival, an improvement on rotarod, normalization and/or improvement in locomotor parameters measured in open field (activity, speed, acceleration, endurance, and gait), a rescue of a 15-20% cortical and striatal neuron loss, normalization of SP striatal neuron neurochemistry, and to a lesser extent enkephalinergic striatal neuron neurochemistry. Deficits were greater in male than female R6/2 mice, and drug benefit tended to be greater in males. The improvements in SP striatal neurons, which facilitate movement, are consistent with the improved movement in LY379268-treated R6/2 mice. Our data indicate that mGluR2/3 agonists may be particularly useful for ameliorating the morphological, neurochemical and motor defects observed in HD.

BDNF may play a differential role in the protective effect of the mGluR2/3 agonist LY379268 on striatal projection neurons in R6/2 Huntington's disease mice.

We have found that daily subcutaneous injection with a maximum tolerated dose (MTD) of the mGluR2/3 agonist LY379268 (20mg/kg) beginning at 4 weeks dramatically improves the phenotype in R6/2 mice. For example, we observed normalization of motor function in distance traveled, speed, the infrequency of pauses, and the ability to locomote in a straight line, and a rescue of a 15-20% striatal neuron loss at 10 weeks. As acute LY379268 treatment is known to increase cortical BDNF production, and BDNF is known to be beneficial for striatal neurons, we investigated if the benefit of daily LY379268 in R6/2 mice for striatal projection neurons was associated with increases in corticostriatal BDNF, with assessments done at 10 weeks of age after daily MTD treatment since the fourth week of life. We found that LY379268 increased BDNF expression in layer 5 neurons in motor cortex, which project to striatum, partly rescued a preferential loss of enkephalinergic striatal neurons, and enhanced substance P (SP) expression by SP striatal projection neurons. The enhanced survival of enkephalinergic striatal neurons was correlated with the cortical BDNF increase, but the enhanced SP expression by SP striatal neurons was not. Thus, LY379268 may protect the two main striatal projection neuron types by different mechanisms, enkephalinergic neurons by the trophic benefit of BDNF, and SP neurons by a mechanism not involving BDNF. The SP neuron benefit may perhaps instead involve the anti-excitotoxic action of mGluR2/3 receptor agonists.

Motor, Visual and Emotional Deficits in Mice after Closed-Head Mild Traumatic Brain Injury Are Alleviated by the Novel CB2 Inverse Agonist SMM-189

We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50–60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50–60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI.