GENSAT BAC cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits

Abstract

Recent development of molecular genetic techniques are rapidly advancing understanding of the functional role of brain circuits in behavior. Critical to this approach is the ability to target specific neuron populations and circuits. The collection of over 250 BAC Cre-recombinase driver lines produced by the GENSAT project provides a resource for such studies. Here we provide characterization of GENSAT BAC-Cre driver lines with expression in specific neuroanatomical pathways within the cerebral cortex and basal ganglia.

Figure 1

The major circuits of the basal ganglia are diagrammed in a sagittal plane. Corticostriatal inputs arise from two major subtypes, intertelencephalic neurons (IT), which provide bilateral inputs to the striatum, and pyramidal tract corticostriatal (PT), which project an axon ipsilatrally, with collaterals to the striatum, thalamus, subthalamic nucleus (STN), superior colliculus, pons, and to the spinal cord. Two main subtypes of projection neurons in the striatum give rise to the direct and indirect pathways. The direct pathway provides direct projections to the output nuclei of the basal ganglia, the internal segment of the globus pallidus (GPi) and the substantia nigra pars reticulata (SNr). The indirect pathway projects to the external segment of the globus pallidus (GPe), which connects indirectly through the STN to the GPi and SNr. The major output of the basal ganglia originates from GABAergic neurons in the GPi and SNr, which provide inhibitory input to the thalamus, superior colliculus and pedunculopontine nucleus (PPN).

Figure 2

BAC-Cre line Rbp4_KL100 with expression in PT and IT corticostriatal neurons. Left column: Sections displaying Cre expression with Rosa26-EGFP reporter at coronal levels through the prefrontal cortex, nucleus accumbens, striatum, thalamus, subthalamic nucleus (STN) and zona incerta (ZI), superior colliculus, and pons. Second column: Injection of AAV-dTomato Cre-dependent expression vector into the secondary motor cortex labels neurons expressing Cre and their axonal projections (pseudo-colored yellow). Labeled axons project bilaterally to the striatum, indicating labeling of IT corticostriatal neurons, and to the ipsilateral thalamus, STN, ZI, superior colliculus and pons, indicting labeling of PT corticostriatal neurons. Injection site and axonal terminal projection areas are shown at progressively higher magnifications in columns to the right.

Figure 3

BAC-Cre lines with select expression in PT corticostriatal neurons. Four BAC-Cre driver lines, Sim1_KJ18, Chrna2_OE25, Efr3a_NO108 and Rcan2_ON50 are described in which AAV-dTomato expression vector injections into primary motor cortex selectively label PT corticostriatal neurons. Cre expression in these lines revealed by crosses to the Rosa26-EGFP reporter line display expression in layer 5 of the cortex, shown in the top row with the inset shown at higher magnification in the second row. AAV-dTomato injection sites into the motor cortex are shown in the third row displaying labeled neurons (pseudo-colored yellow). In each line, axonal projections are distributed to the ipsilateral striatum (caudate-putamen CP), with no evidence of projections to the contralateral hemisphere. Additionally, axonal projections are distributed through the internal capsule with axonal collaterals distributed to the thalamus, subthalamic nucleus (STN), superior colliculus (SC) and pontine nucleus (pons). This pattern of axonal projections is characteristic of pyramidal tract (PT) corticostriatal neurons.

Figure 4

BAC-Cre lines with select expression in IT corticostriatal neurons. Four BAC-Cre lines, Tlx3_PL56, Ebf2_NP183, Sepw1_NP39 and Grp_KH288 are described in which AAV-dTomato expression vector injections into primary and secondary motor cortical areas selectively label intertelencephalic (IT) corticostriatal neurons. Cre-expression revealed by crosses to the Rosa26-EGFP reporter line display expression in layer 5 in neocortical areas in Tlx3_PL56 and Ebf2_NP183, in layer 2/3 in all cortical areas in Sepw1_NP39 and layer 5 and layer 2/3 in secondary motor and prefrontal areas in Grp_KH288 (top 2 rows). AAV-dTomato injection sites label neurons in the cortex, which are shown in the third row (pseudo-colored yellow). In each of these lines, axonal projections are distributed bilaterally within the cerebral cortex and the caudate putamen (CP). Axonal projections from labeled corticostriatal neurons do not extend to the internal capsule, thalamus, subthalamic nucleus (STN), or superior colliculus. This patterns of axonal projections is characteristic of IT corticostriatal neurons.

Figure 5

BAC-Cre lines with expression in prefrontal cortical areas. Four BAC-Cre lines, Colgalt2_NF107, Chrna2_OE25, Sepw_NP39 and Lypd1_NR49 display expression in prefrontal cortical areas revealed by crosses to the Rosa26-EGPF reporter (top row). AAV-dTomato expression vector injections label neurons in the medial prefrontal cortex are shown in the second row, with insets shown at higher magnification in the third row (pseudo-colored yellow). In Colgalt2_NF107, labeled prelimbic cortical neurons in layer 5 provide axonal projections bilaterally to the caudate-putamen (CP) and accumbens (ACC), indicating labeling of IT corticostriatal neurons. Of note, in this case the corticostritatal projections are homogenously distributed to both patch and matrix compartments. Additionally, axonal projections are labeled to the thalamus (not shown), zona incerta (ZI), subthalamic nucleus (STN), superior colliculus (SC) and pons (not shown), indicating labeling of PT corticostriatal neurons. In Chrna2_OE25, a subset of prelimbic layer 5 neurons are labeled, which project to the ipsilateral striatum, thalamus (not shown), ZI, STN, SC and pons (not shown), indicative of selective labeling of PT corticostriatal neurons. In Sepw1_NP39, labled prelimbic neurons in layer 2/3 provide bilateral projections to the CP, but no labeling to the thalamus, ZI, STN, SC or pons, indicating selective labeling of IT corticostriatal neurons. In Lypd1_NR149, labeled neurons in layer 2/3 of the infralimbic cortex, which extend axonal projections to the ipsilateral striatum, but not to the thalamus, STN, ZI, SC or pons, indicating selective labeling of IT corticostriatal neurons. In cases of prefrontal injections in Colgalt2_NF107, Sepw1_NP39 and Lypd1_NR149 there is labeling of axonal projections to the amygdala (amyg), which is not apparent in the injection case in Chrna2_OE25.

Figure 6

BAC-Cre line Syt17_NO14 with expression in reward circuits. Cre-induced expression is demonstrated in BAC-Cre line Syt17_NO14 by crosses to the Rosa26_(CAG)dTomato reporter line in coronal sections at the level of the prefrontal cortex, nucleus accumbens, striatum (caudate putamen, CP), thalamus, amygdala (amyg) and midbrain (pseudocolored green in left hand column). Injection cases of AAV-dTomato expression vectors into areas expressing Cre label neurons (top row) and their axonal projections, shown in column below in matched coronal levels (pseudocolored yellow). Injection case 385 labeled prelimbic cortical neurons, whch provide axonal projections bilaterally to the striatum (caudate putamen, CP), amygdala (amyg), indicative of labeling of IT corticostriatal neurons as well as labeling of projections to the STN, SC and pons, indicative of labeling of PT corticostriatal neurons. In this case there is retrograde labeling of intralaminar thalamic neurons. Injection case 006 labeled intralaminar thalamic neurons, which provide axonal projections to the prelimbic cortex, ipsilateral striatum (caudate putamen, CP and nucleus accumbens (Acc), and to retrosplenial cortical areas. Injection case 356 labeled ventral CA1 hippocampal neurons, which provide axonal projections to the prelimbic cortex, nucleus accumbens (Acc), lateral septal nucleus (LS), hypothalamus (hypo), and amygdala (amyg). Injection case 756 labeled ventral tegmental area dopamine neurons (VTA), which provide axonal projections to the prelimibic cortex, nucleus accumbens (ACC) and ventral caudate putamen (CP), and amygdala (amyg).

Figure 7

BAC-Cre lines with expression in thalamic nuclei. Immunohistochemical Cre-induced EGFP labeling produced by Rosa26_EGFP reporter in 10 lines with expression in various thalamic nuclei. Slc17a6_OX30 (gene also known as Vglut2) has Cre expression in most thalamic nuclei. Other lines show selective expression in thalamic nuclei: Kcnc1_MN77 (MD, VPM, VPL, SMT), Grm2_MR90 (AV,LD, IL, PVT, LH, MH), Gpr26_KO250 (LD, VAL, VM), Vipr2_KH234 (VPM, VPL), Nelf_NR133 (VPM, VPL), Crh_KN282 (VPM, VPL), Gsbs_NL146 (VPM, VPL, SMT), Cnnm2_KD19 (MH), Hap1_RE55 (LH, VM). See table 3 for complete listing.

Figure 8. BAC-Cre lines with preferential expression in Patch/Matrix circuits

Left panels: In lines Plxnd1_OG1 and Colgalt2_NF107, Cre-induced EGFP expression produced with Rosa26_EGFP reporter is shown in the cortex (top row) and striatum (bottom row). Injections of AAV-dTomato expression vectors in Plxnd1_OG1 labels neurons in the somatosensory cortex that provide projections selectively distributed in the striatal matrix compartment (pseudo-colored yellow), whereas injections in Colgalt2_NF107 labels neurons in the prelimbic cortex, which in this case distribute projections to the striatal patch compartment. Right Panels: Striatal patch/matrix projections to the substantia nigra. (top row) Cre-induced EGFP expression produced with Rosa26_EGFP reporter displays expression in neurons selectively in the striatal matrix (Plxnd1_OG1) and patch (Sepw1_NP67) compartments. Injections of AAV-dTomato expression vectors into the striatum in line Pxnd1_OG1 selectively labels neurons in the matrix compartment with axonal projections that terminate in the substantia nigra pars reticulata, which does not contain dopamine neurons (labeled red). In contrast, injections into the striatum in line Sepw1_NP67 labels neurons preferentially in the patch compartment, which provide axonal projections to the substantia nigra that target the dopamine neurons in the pars compacta (labeled red) with some terminals in the pars reticulata.