Selective Vulnerability of Striatal D2 versus D1 Dopamine Receptor-Expressing Medium Spiny Neurons in HIV-1 Tat Transgenic Male Mice

Abstract

Despite marked regional differences in HIV susceptibility within the CNS, there has been surprisingly little exploration into the differential vulnerability among neuron types and the circuits they underlie. The dorsal striatum is especially susceptible, harboring high viral loads and displaying marked neuropathology, with motor impairment a frequent manifestation of chronic infection. However, little is known about the response of individual striatal neuron types to HIV or how this disrupts function. Therefore, we investigated the morphological and electrophysiological effects of HIV-1 trans-activator of transcription (Tat) in dopamine subtype 1 (D1) and dopamine subtype 2 (D2) receptor-expressing striatal medium spiny neurons (MSNs) by breeding transgenic Tat-expressing mice to Drd1a-tdTomato- or Drd2-eGFP-reporter mice. An additional goal was to examine neuronal vulnerability early during the degenerative process to gain insight into key events underlying the neuropathogenesis. In D2 MSNs, exposure to HIV-1 Tat reduced dendritic spine density significantly, increased dendritic damage (characterized by swellings/varicosities), and dysregulated neuronal excitability (decreased firing at 200-300 pA and increased firing rates at 450 pA), whereas insignificant morphologic and electrophysiological consequences were observed in Tat-exposed D1 MSNs. These changes were concomitant with an increased anxiety-like behavioral profile (lower latencies to enter a dark chamber in a light-dark transition task, a greater frequency of light-dark transitions, and reduced rearing time in an open field), whereas locomotor behavior was unaffected by 2 weeks of Tat induction. Our findings suggest that D2 MSNs and a specific subset of neural circuits within the dorsal striatum are preferentially vulnerable to HIV-1.SIGNIFICANCE STATEMENT Despite combination antiretroviral therapy (cART), neurocognitive disorders afflict 30-50% of HIV-infected individuals and synaptodendritic injury remains evident in specific brain regions such as the dorsal striatum. A possible explanation for the sustained neuronal injury is that the neurotoxic HIV-1 regulatory protein trans-activator of transcription (Tat) continues to be expressed in virally suppressed patients on cART. Using inducible Tat-expressing transgenic mice, we found that dopamine subtype 2 (D2) receptor-expressing medium spiny neurons (MSNs) are selectively vulnerable to Tat exposure compared with D1 receptor-expressing MSNs. This includes Tat-induced reductions in D2 MSN dendritic spine density, increased dendritic damage, and disruptions in neuronal excitability, which coincide with elevated anxiety-like behavior. These data suggest that D2 MSNs and specific circuits within the basal ganglia are preferentially vulnerable to HIV-1.

Keywords: Drd1a-tdTomato-expressing neurons; Drd2-eGFP-expressing neurons; basal ganglia; striatal indirect pathway; synaptic dysfunction; whole-cell patch-clamp physiology.

Figure 1.

Structure of D1 and D2 receptor-expressing striatal MSNs in Tat⁻ and Tat⁺ transgenic mice. A–F, D1 (A, C, E) and D2 (B, D, F) MSNs were identified via expression of tdTomato or eGFP, respectively, and filled with biocytin for morphological assessment; a filled axon is shown in E (arrows). C, D, Biocytin-filled neurons were reconstructed in 3D from sequential, Z-stacked, confocal images and their dendritic complexity was analyzed using Bitplane Imaris software (version 7.6.4). G–I, Dendritic complexity (G, H) and overall length (I) as estimated using Scholl analysis did not differ significantly between Tat⁺ and Tat⁻ transgenic mice in either D1 (G) or D2 (H) MSNs. Scale bars: A, B, 10 μm; C, D, 30 μm; and E, F, 5 μm.

Figure 2.

A–J, Synaptodendritic structure of D1 and D2 receptor-expressing striatal MSNs in Tat⁻ and Tat⁺ transgenic mice. No differences in dendritic spine density were observed in D1 MSNs between Tat⁺ and Tat⁻ transgenic mice (A, A′, C); however, Tat⁺ mice had significantly lower dendritic spine density in D2 MSNs compared with Tat⁻ mice (B, B′, D). A′, B′, Superimposed, Bitplane Imaris 3D reconstructions of the same dendrites as in A and B. Scale bar, 3 μm. *Significant main effect of genotype, p < 0.05 (D). E–J, Compared with dendrites of D2 MSNs in Tat⁻ mice (B, I), a significantly greater proportion of dendrites in Tat⁺ mice displayed swellings/varicosities (arrows) and thinning (arrowheads) of the main axis of the dendrite indicative of dendritic damage and/or pending fragmentation (F–H), although dendrites with normal morphology lacking swelling/varicosities could also be found on D2 MSNs (E). Images in E–H were sharpened (Gaussian blur) and median gray levels adjusted in Adobe Photoshop CS6 to better illustrate subtle boundaries, especially those associated with regions of dendritic thinning. Scale bar, 3 μm (F,H are the same scale). *Significant main effect of genotype, p < 0.05 (J).

Figure 3.

A, Depiction of the three classifications of dendritic spines assessed in this study. B–E, Representative dendrite segments from Tat⁻ (B) and Tat⁺ (D) D1 MSNs and from Tat⁻ (C) and Tat⁺ (E) D2 MSNs showing various dendritic spine morphologies. Scale bar, 3 μm. F, G, Percentage of morphologic spine types on D1 MSN dendrites was unchanged between Tat⁺ and Tat⁻ animals (F), whereas the percentage of filopodia/thin-shaped spines on D2 MSN dendrites was decreased in Tat⁺ animals (*p < 0.05), the percentage of stubby spines increased (**p < 0.05), and the proportion of mushroom-shaped spines was unchanged compared with Tat⁻ controls (G).

Figure 4.

A–B′, Representative traces from electrophysiological recordings of D1 (A) and D2 (A′) striatal MSNs at 250 pA (A, A′) or 450 pA (B, B′) of current. C, No differences were observed in the frequency of action potential firing or rheobase (C, inset) in D1 MSNs of mice that expressed the HIV-1 Tat transgene (Tat⁺) or their control counterparts (Tat⁻). C′, However, MSNs in Tat⁺ mice demonstrated a significantly reduced frequency of action potentials while stimulating at 200–350 pA and a significantly greater frequency of action potentials at 450 pA in D2 MSNs compared with Tat⁻ MSNs. D2 MSN rheobase values were significantly greater in Tat⁺ compared with Tat⁻, mice (C′, inset). *Significant difference from Tat⁻ following a significant interaction of genotype and stimulus current (pA) intensity, p < 0.05.

Figure 5.

Anxiety-like and spontaneous motor behavior of equally represented Tat⁺ and Tat⁻ mice crossed to Drd1a-tdTomato or Drd2-eGFP reporter mice. Given that behavior did not differ significantly as a function of the D1 or D2 reporter, analyses of Tat genotype are shown. A–C, In a light–dark transition test, Tat⁺ mice demonstrated significantly reduced latencies to exit the brightly lit chamber (A), made more transitions between the light and dark chambers (B), and demonstrated significantly greater rearing behavior (C) than did Tat⁻ control mice. D, No differences were observed on spontaneous locomotor behavior in an open field as indicated by the total distance traveled. *Significant differences between Tat⁻ and Tat⁺ mice, p < 0.05.