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Comparative density of CCK- and PV-GABA cells within the cortex and hippocampus.

Whissell PD, Cajanding JD, Fogel N, Kim JC - Front Neuroanat (2015)

Bottom Line: However, the relationship and balance between CCK- and PV-GABA neurons in the inhibitory networks of the brain is currently unclear as the distribution of these cells has never been compared on a large scale.The reverse trend was observed for PV-GABA cells.The intersectional genetic labeling approach employed in the current study expands upon the ability to study molecularly defined subsets of GABAergic neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, University of Toronto, Toronto ON, Canada.

ABSTRACT
Cholecystokinin (CCK)- and parvalbumin (PV)-expressing neurons constitute the two major populations of perisomatic GABAergic neurons in the cortex and the hippocampus. As CCK- and PV-GABA neurons differ in an array of morphological, biochemical and electrophysiological features, it has been proposed that they form distinct inhibitory ensembles which differentially contribute to network oscillations and behavior. However, the relationship and balance between CCK- and PV-GABA neurons in the inhibitory networks of the brain is currently unclear as the distribution of these cells has never been compared on a large scale. Here, we systemically investigated the distribution of CCK- and PV-GABA cells across a wide number of discrete forebrain regions using an intersectional genetic approach. Our analysis revealed several novel trends in the distribution of these cells. While PV-GABA cells were more abundant overall, CCK-GABA cells outnumbered PV-GABA cells in several subregions of the hippocampus, medial prefrontal cortex and ventrolateral temporal cortex. Interestingly, CCK-GABA cells were relatively more abundant in secondary/association areas of the cortex (V2, S2, M2, and AudD/AudV) than they were in corresponding primary areas (V1, S1, M1, and Aud1). The reverse trend was observed for PV-GABA cells. Our findings suggest that the balance between CCK- and PV-GABA cells in a given cortical region is related to the type of processing that area performs; inhibitory networks in the secondary cortex tend to favor the inclusion of CCK-GABA cells more than networks in the primary cortex. The intersectional genetic labeling approach employed in the current study expands upon the ability to study molecularly defined subsets of GABAergic neurons. This technique can be applied to the investigation of neuropathologies which involve disruptions to the GABAergic system, including schizophrenia, stress, maternal immune activation and autism.

No MeSH data available.


Related in: MedlinePlus

Selective labeling of CCK- and PV-GABA cells using the dual recombinase–based intersectional genetic strategy. (A) Top. A dual recombinase-responsive reporter allele, RC::FrePe, contains two transcriptional stop cassettes. The first stop cassette is flanked by vertically oriented FRT sites (rectangles, denoted with F) and the other by directly oriented loxP sites (triangles, denoted with P). The loxP-flanked stop cassette also contains mCherry-encoding sequences. Middle: Flpe-mediated stop cassette removal results in mCherry expression. The remaining loxP-flanked stop cassette prevents GFP expression. Bottom: Upon removal of both stop cassettes, requiring Flpe- and Cre-mediated excisions, expression of GFP is turned on and expression of mCherry is turned off. The RC::FrePe allele is knocked-in to the Gt(ROSA)26Sor (R26) locus with CAG (chicken β-actin and CMV enhancer) promoter elements. (B) Venn diagrams illustrating intersectional and subtractive cell populations labeled by the intersectional approach using the RC::FrePe allele. The Dlx5/6-Flpe allele is specific to GABAergic cells in the forebrain. In triple transgenic mice inheriting all three alleles (CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice or PV-Cre;Dlx5/6-Flpe;RC::FrePe mice), cells expressing both Cre and Flpe alleles (i.e., intersectional population) represent CCK- or PV-GABA cells, and are labeled with GFP. In contrast, cells expressing only Flpe (i.e., subtractive population) represent nonCCK-GABA or nonPV-GABA neurons and are labeled with mCherry. (C,D) Specificity of labeling GABA cells using the intersectional genetic strategy. (C) Confocal images of the CA1 stratum radiatum in CCK-Frepe mice (top) and the CA1 stratum pyramidale in PV-Frepe mice (bottom). GFP-expressing cells are labeled in green whereas CCK+ cells (top) and PV+ cells (bottom) are shown in blue. The border between the stratum pyramidale and stratum radiatum is denoted by the dotted line. Scale bar = 20 μm. (D) Percentage of GFP-expressing cells in CCK- and PV-Frepe mice that are CCK+ (orange) or PV+ (blue), respectively. (E) Efficacy of GABA cell labeling in the intersectional genetic approach. Confocal image shows the CA1 stratum radiatum in PV-Frepe mice. GFP- and mCherry-expressing cells are shown in green and red, respectively, whereas GAD67+ cells are shown in blue. Scale bar = 20 μm.
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Figure 1: Selective labeling of CCK- and PV-GABA cells using the dual recombinase–based intersectional genetic strategy. (A) Top. A dual recombinase-responsive reporter allele, RC::FrePe, contains two transcriptional stop cassettes. The first stop cassette is flanked by vertically oriented FRT sites (rectangles, denoted with F) and the other by directly oriented loxP sites (triangles, denoted with P). The loxP-flanked stop cassette also contains mCherry-encoding sequences. Middle: Flpe-mediated stop cassette removal results in mCherry expression. The remaining loxP-flanked stop cassette prevents GFP expression. Bottom: Upon removal of both stop cassettes, requiring Flpe- and Cre-mediated excisions, expression of GFP is turned on and expression of mCherry is turned off. The RC::FrePe allele is knocked-in to the Gt(ROSA)26Sor (R26) locus with CAG (chicken β-actin and CMV enhancer) promoter elements. (B) Venn diagrams illustrating intersectional and subtractive cell populations labeled by the intersectional approach using the RC::FrePe allele. The Dlx5/6-Flpe allele is specific to GABAergic cells in the forebrain. In triple transgenic mice inheriting all three alleles (CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice or PV-Cre;Dlx5/6-Flpe;RC::FrePe mice), cells expressing both Cre and Flpe alleles (i.e., intersectional population) represent CCK- or PV-GABA cells, and are labeled with GFP. In contrast, cells expressing only Flpe (i.e., subtractive population) represent nonCCK-GABA or nonPV-GABA neurons and are labeled with mCherry. (C,D) Specificity of labeling GABA cells using the intersectional genetic strategy. (C) Confocal images of the CA1 stratum radiatum in CCK-Frepe mice (top) and the CA1 stratum pyramidale in PV-Frepe mice (bottom). GFP-expressing cells are labeled in green whereas CCK+ cells (top) and PV+ cells (bottom) are shown in blue. The border between the stratum pyramidale and stratum radiatum is denoted by the dotted line. Scale bar = 20 μm. (D) Percentage of GFP-expressing cells in CCK- and PV-Frepe mice that are CCK+ (orange) or PV+ (blue), respectively. (E) Efficacy of GABA cell labeling in the intersectional genetic approach. Confocal image shows the CA1 stratum radiatum in PV-Frepe mice. GFP- and mCherry-expressing cells are shown in green and red, respectively, whereas GAD67+ cells are shown in blue. Scale bar = 20 μm.

Mentions: To selectively label CCK- and PV-GABA cells for counting, we employed a dual recombinase-based intersectional genetic strategy (Jensen and Dymecki, 2014) (Figure 1). We first crossed the Dlx5/6-Flpe mouse line, which provides a selective genetic access to forebrain GABAergic neurons, with a dual recombinase-responsive reporter line, RC::FrePe (Engleka et al., 2012) (Figure 1A). Subsequently, double transgenic Dlx5/6-Flpe;RC::FrePe mice were crossed with either CCK-Cre or PV-Cre mouse lines, in which the expression of Cre recombinase is restricted to CCK and PV neurons, respectively. The resulting triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice (CCK-Frepe, n = 7) and PV-Cre;Dlx5/6-Flpe;RC::FrePe mice (PV-Frepe, n = 4) were used in cell counting experiments (Figure 1B). In CCK- and PV-Frepe mice, cells expressing Flpe and Cre recombinase (i.e., CCK- or PV-GABA cells) are labeled with reporter enhanced green fluorescent protein (GFP) (Figure 1B). In contrast, cells expressing only Flpe recombinase (i.e., nonCCK-GABA and nonPV-GABA cells) are labeled with the reporter protein mCherry. Cells expressing Cre recombinase alone, or neither recombinase, are unlabeled.


Comparative density of CCK- and PV-GABA cells within the cortex and hippocampus.

Whissell PD, Cajanding JD, Fogel N, Kim JC - Front Neuroanat (2015)

Selective labeling of CCK- and PV-GABA cells using the dual recombinase–based intersectional genetic strategy. (A) Top. A dual recombinase-responsive reporter allele, RC::FrePe, contains two transcriptional stop cassettes. The first stop cassette is flanked by vertically oriented FRT sites (rectangles, denoted with F) and the other by directly oriented loxP sites (triangles, denoted with P). The loxP-flanked stop cassette also contains mCherry-encoding sequences. Middle: Flpe-mediated stop cassette removal results in mCherry expression. The remaining loxP-flanked stop cassette prevents GFP expression. Bottom: Upon removal of both stop cassettes, requiring Flpe- and Cre-mediated excisions, expression of GFP is turned on and expression of mCherry is turned off. The RC::FrePe allele is knocked-in to the Gt(ROSA)26Sor (R26) locus with CAG (chicken β-actin and CMV enhancer) promoter elements. (B) Venn diagrams illustrating intersectional and subtractive cell populations labeled by the intersectional approach using the RC::FrePe allele. The Dlx5/6-Flpe allele is specific to GABAergic cells in the forebrain. In triple transgenic mice inheriting all three alleles (CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice or PV-Cre;Dlx5/6-Flpe;RC::FrePe mice), cells expressing both Cre and Flpe alleles (i.e., intersectional population) represent CCK- or PV-GABA cells, and are labeled with GFP. In contrast, cells expressing only Flpe (i.e., subtractive population) represent nonCCK-GABA or nonPV-GABA neurons and are labeled with mCherry. (C,D) Specificity of labeling GABA cells using the intersectional genetic strategy. (C) Confocal images of the CA1 stratum radiatum in CCK-Frepe mice (top) and the CA1 stratum pyramidale in PV-Frepe mice (bottom). GFP-expressing cells are labeled in green whereas CCK+ cells (top) and PV+ cells (bottom) are shown in blue. The border between the stratum pyramidale and stratum radiatum is denoted by the dotted line. Scale bar = 20 μm. (D) Percentage of GFP-expressing cells in CCK- and PV-Frepe mice that are CCK+ (orange) or PV+ (blue), respectively. (E) Efficacy of GABA cell labeling in the intersectional genetic approach. Confocal image shows the CA1 stratum radiatum in PV-Frepe mice. GFP- and mCherry-expressing cells are shown in green and red, respectively, whereas GAD67+ cells are shown in blue. Scale bar = 20 μm.
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Related In: Results  -  Collection

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Figure 1: Selective labeling of CCK- and PV-GABA cells using the dual recombinase–based intersectional genetic strategy. (A) Top. A dual recombinase-responsive reporter allele, RC::FrePe, contains two transcriptional stop cassettes. The first stop cassette is flanked by vertically oriented FRT sites (rectangles, denoted with F) and the other by directly oriented loxP sites (triangles, denoted with P). The loxP-flanked stop cassette also contains mCherry-encoding sequences. Middle: Flpe-mediated stop cassette removal results in mCherry expression. The remaining loxP-flanked stop cassette prevents GFP expression. Bottom: Upon removal of both stop cassettes, requiring Flpe- and Cre-mediated excisions, expression of GFP is turned on and expression of mCherry is turned off. The RC::FrePe allele is knocked-in to the Gt(ROSA)26Sor (R26) locus with CAG (chicken β-actin and CMV enhancer) promoter elements. (B) Venn diagrams illustrating intersectional and subtractive cell populations labeled by the intersectional approach using the RC::FrePe allele. The Dlx5/6-Flpe allele is specific to GABAergic cells in the forebrain. In triple transgenic mice inheriting all three alleles (CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice or PV-Cre;Dlx5/6-Flpe;RC::FrePe mice), cells expressing both Cre and Flpe alleles (i.e., intersectional population) represent CCK- or PV-GABA cells, and are labeled with GFP. In contrast, cells expressing only Flpe (i.e., subtractive population) represent nonCCK-GABA or nonPV-GABA neurons and are labeled with mCherry. (C,D) Specificity of labeling GABA cells using the intersectional genetic strategy. (C) Confocal images of the CA1 stratum radiatum in CCK-Frepe mice (top) and the CA1 stratum pyramidale in PV-Frepe mice (bottom). GFP-expressing cells are labeled in green whereas CCK+ cells (top) and PV+ cells (bottom) are shown in blue. The border between the stratum pyramidale and stratum radiatum is denoted by the dotted line. Scale bar = 20 μm. (D) Percentage of GFP-expressing cells in CCK- and PV-Frepe mice that are CCK+ (orange) or PV+ (blue), respectively. (E) Efficacy of GABA cell labeling in the intersectional genetic approach. Confocal image shows the CA1 stratum radiatum in PV-Frepe mice. GFP- and mCherry-expressing cells are shown in green and red, respectively, whereas GAD67+ cells are shown in blue. Scale bar = 20 μm.
Mentions: To selectively label CCK- and PV-GABA cells for counting, we employed a dual recombinase-based intersectional genetic strategy (Jensen and Dymecki, 2014) (Figure 1). We first crossed the Dlx5/6-Flpe mouse line, which provides a selective genetic access to forebrain GABAergic neurons, with a dual recombinase-responsive reporter line, RC::FrePe (Engleka et al., 2012) (Figure 1A). Subsequently, double transgenic Dlx5/6-Flpe;RC::FrePe mice were crossed with either CCK-Cre or PV-Cre mouse lines, in which the expression of Cre recombinase is restricted to CCK and PV neurons, respectively. The resulting triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FrePe mice (CCK-Frepe, n = 7) and PV-Cre;Dlx5/6-Flpe;RC::FrePe mice (PV-Frepe, n = 4) were used in cell counting experiments (Figure 1B). In CCK- and PV-Frepe mice, cells expressing Flpe and Cre recombinase (i.e., CCK- or PV-GABA cells) are labeled with reporter enhanced green fluorescent protein (GFP) (Figure 1B). In contrast, cells expressing only Flpe recombinase (i.e., nonCCK-GABA and nonPV-GABA cells) are labeled with the reporter protein mCherry. Cells expressing Cre recombinase alone, or neither recombinase, are unlabeled.

Bottom Line: However, the relationship and balance between CCK- and PV-GABA neurons in the inhibitory networks of the brain is currently unclear as the distribution of these cells has never been compared on a large scale.The reverse trend was observed for PV-GABA cells.The intersectional genetic labeling approach employed in the current study expands upon the ability to study molecularly defined subsets of GABAergic neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, University of Toronto, Toronto ON, Canada.

ABSTRACT
Cholecystokinin (CCK)- and parvalbumin (PV)-expressing neurons constitute the two major populations of perisomatic GABAergic neurons in the cortex and the hippocampus. As CCK- and PV-GABA neurons differ in an array of morphological, biochemical and electrophysiological features, it has been proposed that they form distinct inhibitory ensembles which differentially contribute to network oscillations and behavior. However, the relationship and balance between CCK- and PV-GABA neurons in the inhibitory networks of the brain is currently unclear as the distribution of these cells has never been compared on a large scale. Here, we systemically investigated the distribution of CCK- and PV-GABA cells across a wide number of discrete forebrain regions using an intersectional genetic approach. Our analysis revealed several novel trends in the distribution of these cells. While PV-GABA cells were more abundant overall, CCK-GABA cells outnumbered PV-GABA cells in several subregions of the hippocampus, medial prefrontal cortex and ventrolateral temporal cortex. Interestingly, CCK-GABA cells were relatively more abundant in secondary/association areas of the cortex (V2, S2, M2, and AudD/AudV) than they were in corresponding primary areas (V1, S1, M1, and Aud1). The reverse trend was observed for PV-GABA cells. Our findings suggest that the balance between CCK- and PV-GABA cells in a given cortical region is related to the type of processing that area performs; inhibitory networks in the secondary cortex tend to favor the inclusion of CCK-GABA cells more than networks in the primary cortex. The intersectional genetic labeling approach employed in the current study expands upon the ability to study molecularly defined subsets of GABAergic neurons. This technique can be applied to the investigation of neuropathologies which involve disruptions to the GABAergic system, including schizophrenia, stress, maternal immune activation and autism.

No MeSH data available.


Related in: MedlinePlus