Optogenetic Activation of A11 Region Increases Motor Activity

Limbic brain regions drive goal-directed behaviors. These behaviors often require dynamic motor responses, but the functional connectome of limbic structures in the diencephalon that control locomotion is not well known. The A11 region, within the posterior diencephalon has been postulated to contribute to motor function and control of pain. Here we show that the A11 region initiates movement. Photostimulation of channelrhodopsin 2 (ChR2) transfected neurons in A11 slice preparations showed that neurons could follow stimulation at frequencies of 20 Hz. Our data show that photostimulation of ChR2 transfected neurons in the A11 region enhances motor activity often leading to locomotion. Using vGluT2-reporter and vGAT-reporter mice we show that the A11 tyrosine hydroxylase positive (TH) dopaminergic neurons are vGluT2 and vGAT negative. We find that in addition to dopaminergic neurons within the A11 region, there is another neuronal subtype which expresses the monoenzymatic aromatic L-amino acid decarboxylase (AADC), but not TH, a key enzyme involved in the synthesis of catecholamines including dopamine. This monoaminergic-based motor circuit may be involved in the control of motor behavior as part of a broader diencephalic motor region

Characterization and Behavioural Relevance of the A11 Hypothalamospinal Dopaminergic System in the Mouse

The A11 region, located in the posterior hypothalamus, has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans. It has been suggested to be the primary source of dopaminergic projections to the spinal cord and it may contribute to the control of pain, spinal locomotor network modulation, restless leg syndrome, and cataplexy. However, it remains an understudied dopaminergic nucleus within the brain and we lack understanding of the functional role of this nucleus. My thesis aims to identify and characterize the enzymatic phenotype of A11, its role in locomotor activity and possible connections to other locomotor areas within the brain. I hypothesized that A11 neurons project to the spinal cord and contain the full set of enzymes required for dopamine synthesis. Using immunohistochemistry combined with virus transfection in TH-IRES-Cre mice, I show that A11 neurons (1) directly project to the spinal cord, (2) express tyrosine hydroxylase, as well as AADC, confirming that A11 is dopaminergic, (3) contain vesicular monoamine transporter 2 necessary for packaging DA into vessels but (4) lack the dopamine transporter. I conclude that A11 neurons contain the full complement of enzymes to produce and release dopamine and the lack of DAT could lead to prolonged DA actions within the spinal cord. Furthermore, I hypothesized that the A11 region forms a parallel dopamine-based pathway for movement initiation and control. Using optogenetics, I found that photostimulation of A11 (1) initiates and modulates locomotion in freely moving mice (2) independent of the locus coeruleus. Lastly, I investigate possible direct dopaminergic connections to well known locomotor areas in the brainstem, which could form a parallel pathway to the nigrostriatal system. I found projections from A11 to the medullary reticular formation but not to the mesencephalic locomotor region. Together the data in this thesis reveal a new role for the A11 region in movement control in the freely moving mouse

Characterization of A11 neurons projecting to the spinal cord of mice.

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons

A11 TH positive neurons are also expressing aromatic amino acid decarboxylase (AADC).

<p>Immunohistochemistry targeted against TH (green) and AADC (magenta) shown in a representative middle region section of A11 (A, B) and the locus coeruleus (C). Note the co-localization between TH and AADC positive neurons (arrows) in A11 (B). Locus coeruleus served as a positive control (C). Scale bar 50 µm. A and B are derived from data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109636#pone-0109636-g001" target="_blank">Figure 1</a>. B: Higher magnification of boxed area in A.</p

A11 TH positive neurons project to the spinal cord.

<p>Example of retrograde labelling in the A11 caudal area following FluoroGold (FG) injections into the lumbar spinal cord between lumbar vertebral segments L4 and L5. Representative double-fluorescent immunostaining of tyrosine hydroxylase (TH; green) and FluoroGold (FG; magenta). The white arrows point to double-labeled cells. Most of the TH positive cells also contain FG (arrows), one cell shows labelling for TH but not FG. Several cells that contain FG but not TH are also seen (not co-localizing). Scale bar: 50 µm.</p

YFP expression in A11 neurons following Cre-dependent viral expression and retrograde tracing with FG from the spinal cord.

<p>Rostral region section of A11 from a TH-Cre mouse transduced with a Cre-dependent YFP AAV (green) and immunohistochemistry against FG (magenta) (A–D). Boxed inset in (B (b)) shows FG labeled cells in the motor cortex. Only cells in A11 were co-labeled with YFP and FG (C, D). Scale bars: 50 µm. D: Higher magnification of boxed area in A. Scale bar: 20 µm.</p

Characterization of tyrosine hydroxylase (TH) positive cells in A11.

<p>A: Diagram showing the middle region of the A11 area in the mouse. The red frame represents the area where representative micrographs were taken. The diagram was adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109636#pone.0109636-Paxinos1" target="_blank">[24]</a> with permission. B: TH immunohistochemistry (green) of the middle region of A11. Scale bar 100 µm. 3 V = third ventricle, PF = parafascicular thalamic nucleus, fr = fasciculus retroflexus, PH = posterior hypothalamic nucleus, mt = mammillothalamic tract. C: Diameter of TH positive cells in three regions - rostral (AP −2.0 mm), middle (AP −2.3 mm) and caudal region (AP −2.5 mm). The mean cell diameter was 16.7±0.3 µm and the cell diameters between sections were similar.</p

YFP positive fibers in the lumbar spinal cord originating from A11.

<p>Schematic of fiber localization in the lumbar spinal cord. Representative micrographs of YFP-labeled fibers in transverse (A, B) and parasagittal (C, D) lumbar spinal cord sections. Fibers were found in the dorsal (A) and ventral horn (B) as well as in the grey (C) and the white matter (D). Scale bar: 10 µm.</p