Targeted deletion of Vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse

Figures & data

Figure 1. Specific deletion of Vglut2 in selected forebrain target areas. Floating in situ hybridization on coronal brain (70 μm) sections from control mice and Vglut2^f/f;Emx1-Cre cKO mice (A–H) using a DIG-labelled Vglut2 probe. Close-ups as indicated in I–R, which demonstrate that cells expressing Vglut2 mRNA was absent in the mitral cell (Mi) layer and GI layer in the olfactory bulb (I–J). Vglut2 mRNA was also absent in the RSG of the medial cortex in the Vglut2^f/f;Emx1-Cre mice (K, L). There is a loss of Vglut2 mRNA in the BMA, in the ACo the mRNA is partially deleted, and the Me amygdala and MePV are unaltered in the Vglut2^f/f;Emx1-Cre mice (M–P). Vglut2 mRNA positive cells were present in the Sub in control mice but not in Vglut2^f/f;Emx1-Cre cKO mice (Q, R). ACo = anterior cortical amygdaloid area; BL = basolateral amygdaloid nucleus; BM = basomedial amygdaloid nucleus anterior part; cKO = conditional knock-out; DIG = digoxigenin; GI = periglomerular layer; Me = medial amygdaloid nucleus; MePV = posteriorventral medial amygdaloid nucleus; Mi = mitral cell layer; RSG = retrosplenial group; Sub = subiculum; Bregma interval (dorsal, ventral) is shown in lower right corner.

Table I. Summarized results from in situ hybridization evaluating the presence or not of Vglut2 mRNA in brain structures from three adult Vglut2^f/f;Emx1-Cre cKO mice and three control mice.

Structure		Ctrl	cKO
Pallium	Neocortex	+	–
	RSG	+	–
	Subiculum	+	–
	Hippocampus CA1, CA3, DG	–	–
	Septohippocampus	–	–
	Ventral endopiriform nucleus	+	–*
	Piriform cortex	+	–*
	Claustrum	+	–*
Border regions	Lateral septum	–	–
	Medial septum	–	–
	Rostroventral septum	–	–
	Endopiriform nucleus	–	–
	Lateral amygdala	–	–
	BM	+	–*
	Me	+	+
	ACo	+	–*
Subpallium	Olfactory tuberculum	+	+
	Striatum	–	–
	Ventral pallidum	–	–
	Nucleus accumbens	ND	ND
	Bed nucleus stria terminalis	ND	ND
	Lateral olfactory tract	ND	ND
Olfactory bulb	Mitral cell layer	+	–
	External plexiform	–	–
	Internal plexiform	–	–
	Periglomerular layer	+	–*
	Granule cell	–	–
	Ependymal layer	–	–

The expression for the Emx1-Cre transgene was described previously (Gorski et al. 2002 (31)), and those structures in which Emx1-Cre was there shown to be expressed were analysed for Vglut2 mRNA and listed here.

+ = expression of Vglut2 mRNA Emx1-Cre transgene; – = no expression; –* = low amounts of expression; cKO = conditional knock-out; DG = dentate gyrus; ND = not determined.

Figure 2. Verification that the overall gross anatomy is normal in the Vglut2^f/f;Emx1-Cre mice compared to control mice. Floating in situ hybridization on coronal brain (70 μm) sections from control and Vglut2^f/f;Emx1-Cre cKO mice using a DIG-labelled Viaat (A, B) or Vglut1(C–H) probe. Close-ups show that ACo and BM do not express Vglut1 mRNA, while BL and part of Me express Vglut1 mRNA. ACo = anterior cortical amygdaloid area; BL = basolateral amygdaloid nucleus; BM = basomedial amygdaloid nucleus anterior part; Me = medial amygdaloid nucleus. Bregma interval (dorsal, ventral) is shown in lower right corner.

Figure 3. The Vglut2^f/f;Emx1-Cre mice do not show any altered response to amphetamine. The initial response to novel environment (30 min) and overall response after i.p. injection of saline (90 min), 10 mg/kg, in locomotion, periferal activity, and corner activity are unaltered in the Vglut2^f/f;Emx1-Cre cKO mice compared to control mice (B, D, F). On day 2, the mice were subjected to the activity boxes for 30 min and were thereafter administered 1.5 mg/kg amphetamine i.p. and were recorded for 90 min (A, C, E). The arrows in the graphs depict the time of saline and amphetamine injection. Data were analysed with two-way ANOVA and showed no significant interactions (effect of genotype). Data are represented as mean ± SEM. cKO = conditional knock-out.

Table II. Levels of monoamines and dopamine metabolites (ng/g of wet tissue) of Vglut2^f/f;Emx1-Cre cKO (n = 12) and control (n = 18) mice. The data show no statistically significant alterations between control and cKO mice. Data are presented as mean ± SEM.

Brain region	Metabolite	Ctrl	Vg2^f/f;Emx1-Cre	P value
Nucleus accumbens	Dopamine	0.0129 ± 9.1E-04	0.0122 ± 9.5E-04	0.21
	DOPAC	0.0053 ± 3.9E-04	0.0049 ± 5.7E-04	0.48
	DOPAC/DA	0.4784 ± 3.6E-02	0.4708 ± 4.6E-02	0.99
	NA	0.0015 ± 2.1E-04	0.0015 ± 3.1E-04	0.82
	3-MT	0.0032 ± 2.7E-04	0.0025 ± 2.9E-04	0.26
	5-HT	0.0040 ± 3.1E-04	0.0030 ± 3.8E-04	0.15
Caudatus putamen	Dopamine	0.0197 ± 1.4E-03	0.0200 ± 2.2E-03	0.08
	DOPAC	0.0059 ± 5.2E-04	0.0066 ± 7.0E-04	0.51
	DOPAC/DA	0.3386 ± 4.7E-02	0.2958 ± 2.1E-02	0.47
	NA	0.0004 ± 8.0E-05	0.0003 ± 4.7E-05	0.51
	3-MT	0.0038 ± 5.0E-04	0.0045 ± 4.8E-04	0.36
	5-HT	0.0012 ± 2.2E-04	0.0015 ± 2.1E-04	0.24

P < 0.05 (Mann–Whitney U test).

Nucleus accumbens corresponds to the ventral striatum; Caudatus putamen to the dorsal striatum.

3-MT = 3-methoxytyramine; 5-HT = 5-hydroxytryptamine; cKO, conditional knock-out; DOPAC = 3,4-dihydroxypheneylacetic acid; NA = noradrenalin.

Figure 4. No social deficits or altered despair-like behaviour, but decreased avoidance of open areas and time spent sheltered. Social behaviour analysis on adult Vglut2^f/f;Emx1-Cre cKO mice and control littermates (A, B). The total time spent interacting during a 10-min social interaction session shows no alteration in the Vglut2^f/f;Emx1-Cre cKO mice compared to control mice (A). The number of wins in the social dominance tube test shows that the Vglut2^f/f;Emx1-Cre cKO mice do not display any altered dominance compared to control (B). The Vglut2^f/f;Emx1-Cre mice spent equal time swimming during two 12 min swimming sessions separated 24 h apart in the Porsolt swim test (C). The total activity and the duration in the three different areas in the elevated plus maze show no significant differences between genotypes. The number of total entries in the outer open arm is statistically different between the Vglut2^f/f;Emx1-Cre cKO mice compared to control mice (*P < 0.05, chi-square test) (D). The total activity as measured in the multivariate concentric square field (MCSF) shows no altered behaviour for the Vglut2^f/f;Emx1-Cre cKO mice compared to control mice. The frequency in the central circle and duration in the central circle as well as in central field are significantly increased for the Vglut2^f/f;Emx1-Cre mice compared to control mice; the duration in the dark corner is significantly decreased in the Vglut2^f/f;Emx1-Cre cKO mice compared to control mice (E). Values represent mean ± SEM. * P < 0.05 compared to control littermates. cKO = conditional knock-out.

Gorski JA, Talley T, Qiu M, Puelles L, Rubenstein JL, Jones KR. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J Neurosci. 2002;22:6309–14.

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