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Off-Target Effects in Transgenic Mice: Characterization of Dopamine Transporter (DAT)-Cre Transgenic Mouse Lines Exposes Multiple Non-Dopaminergic Neuronal Clusters Available for Selective Targeting Within Limbic Neurocircuitry

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Off-Target Effects in Transgenic Mice: Characterization of Dopamine Transporter (DAT)-Cre Transgenic Mouse Lines Exposes Multiple Non-Dopaminergic Neuronal Clusters Available for Selective Targeting Within Limbic Neurocircuitry

Maria Papathanou et al. eNeuro.

Abstract

Transgenic mouse lines are instrumental in our attempt to understand brain function. Promoters driving transgenic expression of the gene encoding Cre recombinase are crucial to ensure selectivity in Cre-mediated targeting of floxed alleles using the Cre-Lox system. For the study of dopamine (DA) neurons, promoter sequences driving expression of the Dopamine transporter (Dat) gene are often implemented and several DAT-Cre transgenic mouse lines have been found to faithfully direct Cre activity to DA neurons. While evaluating an established DAT-Cre mouse line, reporter gene expression was unexpectedly identified in cell somas within the amygdala. To indiscriminately explore Cre activity in DAT-Cre transgenic lines, systematic whole-brain analysis of two DAT-Cre mouse lines was performed upon recombination with different types of floxed reporter alleles. Results were compared with data available from the Allen Institute for Brain Science. The results identified restricted DAT-Cre-driven reporter gene expression in cell clusters within several limbic areas, including amygdaloid and mammillary subnuclei, septum and habenula, areas classically associated with glutamatergic and GABAergic neurotransmission. While no Dat gene expression was detected, ample co-localization between DAT-Cre-driven reporter and markers for glutamatergic and GABAergic neurons was found. Upon viral injection of a fluorescent reporter into the amygdala and habenula, distinct projections from non-dopaminergic DAT-Cre neurons could be distinguished. The study demonstrates that DAT-Cre transgenic mice, beyond their usefulness in recombination of floxed alleles in DA neurons, could be implemented as tools to achieve selective targeting in restricted excitatory and inhibitory neuronal populations within the limbic neurocircuitry.

Keywords: Gad1; Vglut2; amygdala; habenula; mammillary; septum.

Figures

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Figure 1.
Figure 1.
Histological analyses using double-in situ hybridization of tdTom and Dat or Th mRNA and immunofluorescence of tdTOM show selective recombination in the ventral midbrain using constitutive and tamoxifen-inducible DAT-Cre mouse lines and identifies ectopic tdTOM-positive neurons in the amygdala. tdTom mRNA in coronal sections of ventral midbrain of tdTomDAT-Cre (A) and (B) tdTomDAT-CreERT2 mice 10 weeks of age. C, D, Double-FISH detecting tdTomato (red) and Dat (green) mRNA in the SNc and subregions of the VTA (IF, PBP, PN, and RLi) of the midbrain. Dat/tdTom mRNA overlap in the SNc and PBP shown in yellow (scale bar 500 μm). Higher magnification of insets of (C’–C’’’) tdTomDAT-Cre and (D’–D’’’) tdTomDAT-CreERT2 mice (scale bar: 25 μm). E, F, Double-FISH for tdTom (red) and Th (green) mRNA in the SNc and VTA with co-localization shown in yellow (scale bar: 500 μm). Higher magnification of insets of (E’–E’’’) tdTomDAT-Cre and (F’–F’’’) tdTomDAT-CreERT2 (scale bar: 25 μm). G, Immunofluorescent coronal section of ventral midbrain showing tdTOM in the SN and VTA but also in BLP (scale bar: 500 μm). tdTom, tdTomato mRNA; tdTOM, tdTomato protein; CA3, CA3 region of hippocampus; BLP, Basolateral amygdaloid nucleus posterior part; Dat, Dopamine transporter; IF, Interfascicular nucleus; PBP, Parabrachial pigmented area; PN, Paranigral nuclei; RLi, Rostral linear nucleus; SNc, Substantia nigra pars compacta; SNr, Substantia nigra pars reticulata; tdTom, tdTomato mRNA; tdTOM, tdTomato protein; Th, Tyrosine hydroxylase; VTA: Ventral tegmental area; CA3, CA3 region of hippocampus.
Figure 2.
Figure 2.
Immunohistofluorescent analysis of brains derived from tdTomDAT-Cre and tdTomDAT-CreERT2 mice identifies tdTOM-positive cell bodies in the LS, LHb, and the amygdala. Series of coronal sections from the level of the striatum and NAc through to the raphe nucleus showing expression pattern of recombination in (A–G) tdTomDAT-Cre and (H–N) tdTomDAT-CreERT2 mice (scale bar: 2 mm). Expression of tdTOM-positive neurons in the (O, T) lateral septum, (P, U) LHb, (Q, V) BLP, (R, W) arcuate nucleus, and (S, X) the premammilary nucleus of the hypothalamus in tdTomDAT-Cre and tdTomDAT-CreERT2 mice (scale bar: 100 μm). ADP, anterodorsal preoptic nucleus; Arc, arcuate nucleus; CPu, caudate putamen; DR, dorsal raphe; Hyp, hypothalamus; LS, lateral septum; MFB, medial forebrain bundle; MHb, medial habenula; PAG, periaquaductal gray; Pe, periventricular hypothalamic nucleus; STN, subthalamic nucleus; ZI, zona incerta; BLP, Basolateral amygdaloid nucleus posterior division; BNST, Bed nucleus of the stria terminalis; CeA, Central amygdala, CLi, Caudal linear nucleus; NAc, Nucleus accumbens; MePD, Medial amygdaloid nucleus posterodorsal part, and LHb, Lateral habenula, PMH, Premammillary nucleus; RMN, Retromammillary nucleus; RRF, Retrorubral field; SNc, Substantia nigra pars compacta; SNr, Substantia nigra pars reticulata; VTA: Ventral tegmental area.
Figure 3.
Figure 3.
Clusters of mCHERRY-positive cell bodies confirm findings observed with tdTOM. Expression of mCHERRY-positive cell bodies in combination with (A–G) TH and (H–N) DAT immunofluorescence in the (A, H) midbrain: VTA and SNc (B, I) the arcuate nucleus and (C, J) the PMH (D, K) the lateral septum (E, L) the LHb (F, M) the BLP and (G, N) the RMN of mCherryTRAPDAT-Cre mice (white arrows showing mCHERRY-positive cell bodies, green arrows indicating TH-positive cells, and yellow arrows illustrating examples of co-localization; scale bar: 50 μm). See also Extended Data Figure 3-1. Arc, arcuate nucleus; CPu, caudate putamen; D3V, dorsal third ventricle; LS, lateral septum; LV, lateral ventricle; MHb, medial habenula; 3V, third ventricle Arc, Arcuate nucleus; CPu, Caudate putamen; D3V, Dorsal 3rd ventricle; LHb, Lateral habenula; LS, Lateral septum; LV, Lateral ventricle; BLP, Posterior part of basolateral amygdala; MHb, Medial habenula; PMH, Premammillary nucleus; RMN; Retromammillary; SNc, Substantia nigra pars compacta; VTA: Ventral tegmental area; 3V, Third ventricle.
Figure 4.
Figure 4.
A subset of mCHERRY-positive cell bodies in the midbrain, the LHb, the PMH, and the RMN are immuno-positive for CALBINDIN (CALB1) and CALRETININ (CALB2). Immunofluorescent histological analysis of (A–G) mCHERRY (red) and CALB1 (green) and (H–N) mCHERRY (red) and CALB2 (green) in the (A, H) midbrain, (B, I) arcuate nucleus, and (C, J) premammillary of the hypothalamus (PMH) (D, K), the lateral septum, (E, L) LHb, (F, M) BLP of the amygdala, and (G, N) RMN (yellow arrows illustrate example of cells where co-localization is detected; scale bar: 50 μm). Arc, arcuate nucleus; D3V, dorsal third ventricle; LS, lateral septum; MHb, medial habenula; PV, paraventricular thalamic nucleus; 3V, third ventricle; Arc, Arcuate nucleus; BLP, Basolateral amygdaloid nucleus posterior division; D3V, Dorsal third ventricle; LHb, Lateral habenula; LS, Lateral septum; MHb, Medial habenula; PMH, Premammillary nucleus of the hypothalamus; PV, Paraventricular thalamic nucleus; RMN, Retromammillary; SNc, Substantia nigra pars compacta; VTA: Ventral tegmental area; 3V, Third ventricle.
Figure 5.
Figure 5.
Double-FISH analyses of tdTom mRNA with Dat, Th, Vglut2, and Gad1 mRNAs identify multiple sites of tdTom/Vglut2 and tdTom/Gad1 double-positive cells in the brain of tdTomDAT-Cre and tdTomDAT-CreERT2 mice. Double-FISH analysis of (A) tdTom and Vglut2 mRNAs or (B) tdTom and Gad1 mRNAs in the midbrain of tdTomDAT-Cre mice and (J, K) tdTomDAT-CreERT2 mice. Higher magnification of tdTom/Dat, tdTom/Th, tdTom/Vglut2, and tdTom/Gad1 mRNA within (C–C’’’) the VTA and SN (D–D’’’) the arcuate nucleus (E–E’’’) and the PMH, (F’–F’’’) in the lateral septum (G–G’’’) the LHb, (H–H’’’) the basal amygdala, (I–I’’’) and the RMN of tdTomDAT-Cre mice. L–T’’’, Similar expression analysis was performed in the tdTomDAT-CreERT2 mice for (L–T) tdTom/Dat, (L’–T’) tdTom/Th, (L’’–T’’) tdTom/Vglut2, and (L’’’–T’’’) tdTom/Gad1 mRNA in the different regions (yellow arrows indicate examples of colocalization between tdTom and Dat, Th, Vglut2, or Gad1 mRNA; scale bars: 275 and 150 μm; insets: 50 μm). Arc, arcuate nucleus; LS, lateral septum, MHb, medial habenula; SN, substantia nigra; Arcuate nucleus; BLP, Basolateral amygdaloid nucleus posterior division; LHb, Lateral habenula; LS, Lateral septum, MHb, Medial habenula; PMH, Premammillary nucleus of the hypothalamus, RMN, Retromammillary nucleus; SN, Substantia nigra, SNc, Substantia nigra pars compacta; VTA, Ventral tegmental area.
Figure 6.
Figure 6.
Intracranial injection of AAV5-eYFP into the VTA of tdTomDAT-Cre mice verifies activity of Cre recombinase and enables identification of projections. A, A’, B, B’, Stereotaxic viral injections of AAV5-EF1a-DIO-eYFP into the VTA of tdTomDAT-Cre mice. Schematic illustration shows the injection site (A’). eYFP (A–D); eYFP and tdTOM (A’–D’). A, A’ and B, B’, Two different rostro-caudal levels of the midbrain. Cytoplasmic eYFP (green) detected in the VTA and some also in the SNc (A, B) with eYFP-positive projections detected in the BLP (in the section level shown in A). tdTOM-positive cells in VTA and SNc (A’, B’); also, the tdTOM-positive cell cluster shown in Figure 1 is detected in the BLP at the section level of A (A’; inset showing tdTOM-positive cell cluster and eYFP-positive fibers in the BLP, A’). C, C’, D, D’, eYFP-positive projections in additional target structures known for the VTA and SNc: the BLA and CeA of the amygdala and the habenula (C–C’), and the dorsal and ventral aspects of the striatum (D–D’). At this section level, TdTOM is detected in the CeA (scale bar: 1.25 mm; inset: 100 μm). See also Extended Data Figure 6-1. BLA, basolateral amygdala; CA1, CA1 region of hippocampus; cc, corpus callosum; CPu, caudate putamen; IPN, interpeducular nucleus; LS, lateral septum; LV, lateral ventricle; MFB, medial forebrain bundle; MHb, medial habenula; NAcSh, NAc shell; OT, olfactory tubercle; VTAr, VTA rostral part.
Figure 7.
Figure 7.
Intracranial injections of AAV5-eYFP into the LHb and into the amygdala of tdTomDAT-Cre mice verify Cre activity in these areas, and enable identification of distinct projections from each of these areas. A, Stereotaxic injections with AAV5-EF1a-DIO-eYFP into the LHb of tdTomDAT-Cre mice. eYFP (green) and tdTOM (red) immunofluorescent-positive cell bodies of the LHb; eYFP (green)-positive projections detected in (B) the medioventral VTA, and (C) in the median raphe nucleus. D, Stereotaxic injections with AAV5-EF1a-DIO-eYFP into the LHb of tdTomDAT-Cre mice. eYFP (green) and tdTOM (red) immunofluorescent-positive cell bodies in the BLP of amygdala and eYFP-positive projections in (E) the ventral striatum (NAcC) and (F) the BNST (scale bar: 50 μm; inset: 25 and 40 μm). Aca, anterior commissure, anterior part; LV, lateral ventricle; MHb, medial habenula; MnR, medial raphe nucleus, NAcSh, NAc shell; SN, substantia nigra.
Figure 8.
Figure 8.
Schematic drawing illustrating projections from amygdala and LHb seen by viral-genetic tracing of non-dopaminergic DAT-Cre neurons identified in the study. VTA injections used as control. CPu, caudate putamen; LV, lateral ventricle; NAcSh, NAc shell; OB, olfactory bulb; PFC, prefrontal cortex; RN, raphe nucleus; SN, substantia nigra Hyp, Hypothalamus; NAcC, NAc core.

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