Noradrenergic fibers innervate the entire cerebral cortex, whereas the cortical distribution of dopaminergic fibers is more restricted. However, the relative functional impact of noradrenalin and dopamine receptors in various cortical regions is largely unknown. Using a specific genetic label, we first confirmed that noradrenergic fibers innervate the entire cortex whereas dopaminergic fibers were present in all layers of restricted medial and lateral areas but only in deep layers of other areas. Imaging of a genetically encoded sensor revealed that noradrenalin and dopamine widely activate PKA in cortical pyramidal neurons of frontal, parietal and occipital regions with scarce dopaminergic fibers. Responses to noradrenalin had higher amplitude, velocity and occurred at more than 10-fold lower dose than those elicited by dopamine, whose amplitude and velocity increased along the antero-posterior axis. The pharmacology of these responses was consistent with the involvement of Gs-coupled beta1 adrenergic and D1/D5 dopaminergic receptors, but the inhibition of both noradrenalin and dopamine responses by beta adrenergic antagonists was suggestive of the existence of beta1-D1/D5 heteromeric receptors. Responses also involved Gi-coupled alpha2 adrenergic and D2-like dopaminergic receptors that markedly reduced their amplitude and velocity and contributed to their cell-to-cell heterogeneity. Our results reveal that noradrenalin and dopamine receptors both control cAMP-PKA signaling throughout the cerebral cortex with moderate regional and laminar differences. These receptors can thus mediate widespread effects of both catecholamines, which are reportedly co-released by cortical noradrenergic fibers beyond the territory of dopaminergic fibers.
The catecholamines dopamine (DA) and noradrenalin (NA) are neurotransmitters that widely modulate brain circuits and behaviors. NA is involved in arousal, attention, memory, and stress whereas DA is implicated in learning, reward, attention, and movement control. Likewise, catecholaminergic dysfunctions are associated with cognitive, emotional, and motor disorders and catecholaminergic transmission is the target of multiple drugs used in therapy of human brain disorders. Catecholamines are synthesized in discrete brainstem nuclei via a common pathway involving tyrosine hydroxylase (TH) that leads to DA production, which is converted to NA by DA beta hydroxylase (DBH). The effects of DA and NA are mediated by G protein-coupled receptors. The five DA receptors belong to the D1/D5 or D2-like classes, which activate or inhibit cAMP/protein kinase A (PKA) signaling via Gs or Gi proteins, respectively. NA also regulates the cAMP/PKA pathway by activating Gs-coupled beta1–3 receptors or Gi-coupled alpha2 adrenoceptors. NA additionally activates the phospholipase C pathway through Gq-coupled alpha1 receptors.
Dopamine- and NA-containing fibers exhibit wide, but distinctive, distributions in the brain. Catecholaminergic projections to the cerebral cortex stem from DA neurons of the ventral tegmental area (VTA) and NA neurons of the locus coeruleus (LC). LC fibers innervate the entire cortical mantle, whereas VTA fibers distribute in all layers of medio-frontal and ventro-lateral cortices, but are restricted to deep layers in other areas of the rodent cortex. The broad distribution of NA receptors in the rodent cortex is consistent with that of LC fibers. In contrast, a mismatch exists between the widespread expression also reported for DA receptors, and the restricted distribution of VTA fibers in the rodent cortex. While LC fibers are a plausible source of cortical DA outside the VTA projection areas, the question of the relative functional impact of DA and NA receptors in various cortical regions has not been addressed.
In the present study, we first examined the distribution of LC and VTA fibers in the rodent cortex using site and cell-type-specific labeling of catecholaminergic neurons with green fluorescent protein (GFP) via conditional viral transfer. We then characterized the functional impact of Gs- and Gi-coupled DA and NA receptors on cAMP/PKA signaling in layers II/III and V of the frontal, parietal, and occipital cortex using 2-photon imaging of a genetically encoded PKA sensor in rat brain slices. Our results confirm the differential distribution of LC and VTA fibers in the cortex and reveal that both NA and DA receptors control cAMP-PKA signaling throughout the cerebral cortex with moderate regional and laminar differences.
All the experiments were performed according to the guidelines of the French Ministry of Agriculture, Food Processing Industry and Forestry for handling animals (decree 2013-118). Transgenic DBH-Cre mice (DBH-cre) were a gift from Bruno Giros McGill University, Canada, MMRRC line: Tg(Dbh-cre) KH212Gsat/Mmucd, stock number 032081-UCD. DA transporter-Cre mice (DAT-cre) were a gift from Uwe Maskos Institut Pasteur, France, (Tg)BAC-DATiCrefto. Male Wistar rats (12–15 days old) were obtained from Janvier Labs. Animals were maintained in a 12 h light–12 h dark cycle, in stable conditions of temperature (22°C), with food and water available ad libitum.
Site- and cell-type-specific labeling of NA or DA neurons was achieved by stereotactic injection of a viral vector into the LC of DBH-Cre or the VTA of DAT-Cre mice (1–3 month-old). The viral vector was a recombinant adeno-associated virus (AAV) driving Cre-dependent expression of a fusion protein containing channelrhodopsin 2 (ChR2) and a yellow variant (YFP) of the GFP from Aequorea victoria. The Cre-inducible vector AAV2/1-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-HGHpA (titer: 3 × 10 11 gc/ml) was produced from Addgene plasmid #20298 at the vector core facility of Nantes University (UMR 1089 IRT1, France). Aliquots of the pseudovirion were stored at -80°C before stereotactic injection. Mice (six DAT-Cre and six DBH-Cre) were anesthetized with isoflurane and placed on a small animal stereotactic frame. For transduction of LC NA neurons, AAV-EF1α-DIO-ChR2-YFP pseudovirion was bilaterally injected adjacent to the LC at coordinates from bregma: antero-posterior (AP), -5.45 mm; medio-lateral (ML), ±1 mm; dorso-ventral (DV) -3.65 mm). For DA neuron transduction, pseudovirion was bilaterally injected into the VTA (AP: -3.4 mm, ML: ±0.5 mm, DV: -4.4 mm). Injections were performed through an internal canula at a rate of 0.1 μl/min for 10 min (total 1 μl per site). The canula was held in place for 15 min before retraction out of the brain.
Eight weeks after injection, mice were deeply anesthetized with 10 mg/ml ketamine and 0.1% xylazine before transcardiac perfusion with 50 ml of 4% paraformaldehyde in 0.12 mM phosphate buffer (pH 7.4). Brains were removed and post-fixed with the same solution for 2 h, cryoprotected with 30% sucrose and cut with a freezing microtome (Leica) at a thickness of 40 μm. Slices were washed overnight with PBS, then blocked and permeabilized with PBS complemented with 0.2% fish skin gelatin, 0.25% triton X-100 (PBS-GT) for 2 h at room temperature. Brain slices were incubated overnight at 4°C with primary antibody diluted in PBS-GT (monoclonal mouse anti TH: MAB318 (Clone LNC1), Millipore, 1/2000; polyclonal chicken anti-GFP: GFP-1020 (Aves Labs, 1/2000). After 6 ×10 min washes in PBS-GT, slices were incubated for 3 h at room temperature with fluorescently labeled secondary antibody diluted at 1/1000 in PBS-GT (goat anti chicken alexa488, Invitrogen A11039; goat anti mouse Alexa555, Invitrogen A21422). Slices were mounted in fluoromount (Clinisciences) after extensive washes in PBS-GT followed by PBS. Images were obtained using an AXIO Zoom.V16 macroscope (Zeiss). Labeled fibers were drawn in black (value of the pixel: 255) on white background (value of the pixel: 0) using Image J and Photoshop Cs2 (Adobe). The density of fibers was obtained from drawings using the line plot profile tool of ImageJ on lanes of 200 pixels (260 μm) width covering the vertical extent of the cortex from pia to white matter. The fiber density corresponds to the averaged value of black and white pixels in a 200 pixel line projected on the vertical axis along the lane.
Rats were killed by decapitation. Brains were quickly removed and immersed in ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4, 2 CaCl 2, 1 MgCl 2, 26 NaHCO 3, 20 D-glucose, 5 Na pyruvate, 1 kynurenic acid, and saturated with 5% CO 2/95% O 2. Parasagittal slices (300 μm thick) of cortex were cut at an angle of 10° using a Leica VT 1000S Vibratome (Leica). Slices were kept at room temperature for 30 min in the same solution. Brain slices were placed onto a millicell-CM membrane (Millipore) with culture medium (50% minimum essential medium, 50% Hanks’ balanced salt solution, 6.5 g/L glucose, and 100 U/ml penicillin/100 μg/ml streptomycin; Invitrogen). Transduction was performed by adding ∼5 × 10 5 particles per slice of sindbis virus encoding the GAkdYmut sensor that reports PKA activation through a reversible increase of fluorescence intensity. Slices were incubated overnight at 35°C in 5% CO 2. The next morning, brain slices were equilibrated in ACSF for 1 h and then placed into the recording chamber and perfused continuously at 2 ml/min with ACSF at 32°C. Under these conditions, sindbis viral transduction efficiently and selectively targets cortical pyramidal neurons, and leaves their functional properties essentially unaffected.
Two-photon images were obtained with a custom-built 2-photon laser scanning microscope as described, based on an Olympus BX51WI upright microscope (Olympus, Tokyo, Japan) with ×40 (0.8 NA) or ×60 (0.9 NA) water-immersion objectives and a titanium:sapphire laser (MaiTai HP; Spectra Physics, Ellicot City, MD, USA). Two-photon excitation was performed at 920 nm for GFP. Images were acquired as z stacks and analyzed using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA). Occasional x, y, and z drifts were corrected using custom macros developed from ImageJ plugins TurboReg, StackReg, MultiStackReg, and Image CorrelationJ. Fluorescence variations were measured at the soma of pyramidal neurons. Fluorescence intensity of regions of interest (ROIs) was calculated for each time point from average intensity projection of 3–5 frames by averaging pixel intensity. Variations of fluorescence intensity in a given ROI were expressed as the ratio Δ F/F 0 and calculated according to the formula (F - F 0)/F 0. F corresponds to the fluorescence intensity in the ROI at a
