Motor slowing, forebrain white matter loss, and striatal shrinkage have been reported in premanifest Huntington's 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.

Brain-derived neurotrophic factor (BDNF) plays important roles in the development, maintenance, and plasticity of the mammalian forebrain. These functions include regulation of neuronal maturation and survival, axonal and dendritic arborization, synaptic efficacy, and modulation of complex behaviors including depression and spatial learning. Although analysis of mutant mice has helped establish essential developmental functions for BDNF, its requirement in the adult is less well documented. We have studied late-onset forebrain-specific BDNF knockout (CaMK-BDNF(KO)) mice, in which BDNF is lost primarily from the cortex and hippocampus in early adulthood, well after BDNF expression has begun in these structures. We found that although CaMK-BDNF(KO) mice grew at a normal rate and can survive more than a year, they had smaller brains than wild-type siblings. The CaMK-BDNF(KO) mice had generally normal behavior in tests for ataxia and anxiety, but displayed reduced spatial learning ability in the Morris water task and increased depression in the Porsolt swim test. These behavioral deficits were very similar to those we previously described in an early-onset forebrain-specific BDNF knockout. To identify an anatomical correlate of the abnormal behavior, we quantified dendritic spines in cortical neurons. The spine density of CaMK-BDNF(KO) mice was normal at P35, but by P84, there was a 30% reduction in spine density. The strong similarities we find between early- and late-onset BDNF knockouts suggest that BDNF signaling is required continuously in the CNS for the maintenance of some forebrain circuitry also affected by developmental BDNF depletion.

Brain-derived neurotrophic factor (BDNF), a neurotrophin, enhances the survival and differentiation of several classes of neurons in vitro. To determine its essential functions, we have mutated the BDNF gene. Most homozygote mutants die within 2 days after birth, but a fraction live for 2-4 weeks. These develop symptoms of nervous system dysfunction, including ataxia. The BDNF mutant homozygotes have substantially reduced numbers of cranial and spinal sensory neurons. Although their central nervous systems show no gross structural abnormalities, expression of neuropeptide Y and calcium-binding proteins is altered in many neurons, suggesting they do not function normally. In contrast with mice lacking the BDNF receptor TrkB, motor neurons appear normal in the BDNF mutant.

We evaluated the impact of early embryonic deletion of huntingtin (htt) from pyramidal neurons on cortical development, cortical neuron survival and motor behavior, using a cre-loxP strategy to inactivate the mouse htt gene (Hdh) in emx1-expressing cell lineages. Western blot confirmed substantial htt reduction in cerebral cortex of these Emx-httKO mice, with residual cortical htt in all likelihood restricted to cortical interneurons of the subpallial lineage and/or vascular endothelial cells. Despite the loss of htt early in development, cortical lamination was normal, as revealed by layer-specific markers. Cortical volume and neuron abundance were, however, significantly less than normal, and cortical neurons showed reduced brain-derived neurotrophic factor (BDNF) expression and reduced activation of BDNF signaling pathways. Nonetheless, cortical volume and neuron abundance did not show progressive age-related decline in Emx-httKO mice out to 24months. Although striatal neurochemistry was normal, reductions in striatal volume and neuron abundance were seen in Emx-httKO mice, which were again not progressive. Weight maintenance was normal in Emx-httKO mice, but a slight rotarod deficit and persistent hyperactivity were observed throughout the lifespan. Our results show that embryonic deletion of htt from developing pallium does not substantially alter migration of cortical neurons to their correct laminar destinations, but does yield reduced cortical and striatal size and neuron numbers. The Emx-httKO mice were persistently hyperactive, possibly due to defects in corticostriatal development. Importantly, deletion of htt from cortical pyramidal neurons did not yield age-related progressive cortical or striatal pathology.

Striatal degeneration in Huntington's disease (HD) is associated with increases in perikaryal calbindin immunolabeling in yet-surviving striatal projection neurons. Since similar increases have also been observed in surviving striatal projection neurons after intrastriatal injection of the excitotoxin quinolinic acid, the increased calbindin in HD striatum has been interpreted to suggest an excitotoxic process in HD. We used immunolabeling to assess if calbindin is elevated in striatal projection neurons of R6/2 HD transgenic mice. These mice bear exon 1 of the human huntingtin gene with 144 CAG repeats and show some of the neuropathological signs (e.g., neuronal intranuclear inclusions) and clinical traits (e.g., wasting prior to early death) of HD. We found an increased frequency of calbindin-immunoreactive neuronal perikarya in the striatum of 6- and 12-week-old R6/2 mice compared to wild-type controls. This increase was most notable in the normally calbindin-poor dorsolateral striatum. We found no significant changes in the total area of striatum occupied by the calbindin-negative striosomes and no consistent changes in striatal calbindin mRNA. The increase in calbindin in R6/2 striatal neurons was thus limited to the matrix compartment, and it may be triggered by increased Ca2+ entry due to the demonstrated heightened NMDA sensitivity of these neurons. The data further support the similarity of R6/2 mice to HD, and are consistent with the occurrence of an excitotoxic process in striatum in both.

Excitotoxic injury to striatum by dysfunctional cortical input or aberrant glutamate uptake may contribute to Huntington's 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.

To obtain insights into the expression of neurotrophin-3 (NT-3) in the mouse, we have utilized mice in which the Escherichia coli lacZ gene is integrated into the neurotrophin-3 locus (NT-3(lacZneo), Fariñas et al. [1994] Nature 369:658-661). In this mouse strain, beta-galactosidase production is under control of the NT-3 promoter in its normal chromosomal environment, and histochemical measurement of beta-galactosidase provides a simple, sensitive method to determine which cells express NT-3. Our data correlate well with previous in situ mRNA and immunocytochemical studies reporting the localization of NT-3. For example, in adult NT-3(lacZneo)/+ mice, beta-galactosidase is expressed in high amounts in limbic areas of the cortex (cingulate, retrosplenial, piriform, and entorhinal), in the visual cortex, in the hippocampal formation (dentate granule cells, CA2 cells, fasciola cinereum, induseum griseum, tenia tecta, presubiculum, and parasubiculum), and in the septum (septohippocampal nucleus and lateral dorsal septum). In contrast with other studies, our results suggest more extensive expression of NT-3 in adult and aged mouse brains with cortical expression apparent at 4.5 months. To further define the cell populations expressing NT-3 in adult mice, we have combined immunocytochemistry with histochemical staining and found that beta-galactosidase is coexpressed with a neuronal marker (NeuN) and with parvalbumin and neuropeptides, markers for GABAergic interneurons. Our studies of embryonic beta-galactosidase expression in NT-3(lacZneo)/+ mice suggest sites of NT-3 expression not previously described, including embryonic piriform cortical cells and dentate granule cell precursors.

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.

Prior studies suggest differences exist among striatal projection neuron types in their vulnerability to Huntington's disease (HD). In the present study, we immunolabeled the fibers and terminals of the four main types of striatal projection neuron in their target areas for substance P, enkephalin, or glutamic acid decarboxylase (GAD), and used computer-assisted image analysis to quantify the abundance of immunolabeled terminals in a large sample of HD cases ranging from grade 0 to grade 4 [J. Neuropathol. Exp. Neurol. 44 (1985) 559], normalized to labeling in control human brains. Our goal was to characterize the relative rates of loss of the two striatopallidal projection systems (to the internal versus the external pallidal segments) and the two striatonigral projections systems (to pars compacta versus pars reticulata). The findings for GAD and the two neuropeptides were similar--the striatal projection to the external pallidal segment was the most vulnerable, showing substantial loss by grade 1. Loss of fibers in both subdivisions of the substantia nigra was also already great by grade 1. By contrast, the loss in the striatal projection system to the internal segment of globus pallidus proceeded more gradually. By grade 4 of HD, however, profound loss in all projection systems was apparent. These findings support the notion that the striatal neurons preferentially projecting to the internal pallidal segment are, in fact, less vulnerable in HD than are the other striatal projection neuron types.

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.