Neuronal NT-3 is not required for synaptic transmission or long-term potentiation in area CA1 of the adult rat hippocampus

Abstract

Neurotrophic factors, including BDNF and NT-3, have been implicated in the regulation of synaptic transmission and plasticity. Previous attempts to analyze synaptic transmission and plasticity in mice lacking the NT-3 gene have been hampered by the early death of the NT-3 homozygous knockout animals. We have bypassed this problem by examining synaptic transmission in mice in which the NT-3 gene is deleted in neurons later in development, by crossing animals expressing the CRE recombinase driven by the synapsin I promoter to animals in which the NT-3 gene is floxed. We conducted blind field potential recordings at the Schaffer collateral-CA1 synapse in hippocampal slices from homozygous knockout and wild-type mice. We examined the following indices of synaptic transmission: (1) input-output relationship; (2) paired-pulse facilitation; (3) post-tetanic potentiation; and (4) long-term potentiation: induced by two different protocols: (a) two trains of 100-Hz stimulation and (b) theta burst stimulation. We found no difference between the knockout and wild-type mice in any of the above measurements. These results suggest that neuronal NT-3 does not play an essential role in normal synaptic transmission and some forms of plasticity in the mouse hippocampus.

Figure 1

Strategy for generating NT-3 conditional knockout mice. (a) Genomic structure of mouse NT-3 gene. (b) Homologous recombinant allele. Targeting vector was transfected into CJ7 ES cells and the details of selection were as described previously (Tessarollo et al. 1994). Homologous recombination was screened with Southern blot using both 5′ and 3′ probes. (c) Generation of cko⁺ allele. Recombinant ES clones were transfected with a CMV–Cre-expressing plasmid to delete the TK-neo selection cassette (Kuhn et al. 1995; Ramirez-Solis et al. 1995). Southern blot was carried out to screen for NT-3 allele (NT-3–loxp allele) flanked by two loxp sites. ES clones with NT-3–loxp were injected into blastocysts for germ-line transfer. (d) Generation of cko⁻allele. Mice with NT-3–loxp allele were then crossed with Synapsin I–Cre (SynI–Cre) (Schoch et al. 1996) transgenic mice to produce NT-3 conditional knockout mice with cko allele. From a–d, restriction sites are (B) BamHI, (E) EcoRI.

Figure 2

Characterization of SynI–Cre, NT-3 conditional knockout mice. (a) PCR assay from tissue genomic DNAs indicating the appearance in brain of Cre-specific NT-3–loxp (cko⁻) allele (see arrowhead) indicating tissue specific cre-mediated looping out of the NT-3 coding exon. (b) Southern blot of whole brain genomic DNA to show the extent of the NT-3 deletion by SynI–Cre to transform the cko⁺ allele into cko⁻. (c) Approximately 63% of the whole brain genomic cko⁺ allele was transformed into cko⁻ NT-3 gene by SynI–Cre. (d) Northern blot analysis of whole brain RNA with NT-3 probe to show the decrease of NT-3 mRNA after exposure to SynI–Cre. GAPDH mRNA was used as an internal loading control. (e) Quantification of NT-3 mRNA levels with PhosphorImager indicates a 75% reduction brain NT-3 mRNA. (f,g) SynI–Cre mice were crossed with a lacZ reporter mouse strain (Tsien et al. 1996) to show the regions of Cre expression, which was recapitulated by lacZ expression. (g) Higher magnification of hippocampal region of f. (f) Bar, 1000 μm; (g) bar, 250 μm.

Figure 3

Input–output relations are normal in hippocampal slices from conditional neuronal NT-3 mutant mice. Shown are the mean fEPSP responses to stimuli of increasing strengths for both the knockout (SynI–cre/cko⁻) and control (cko⁺/cko⁺) slices. There was no significant difference between the two groups. (n) Number of slices. The number of animals from which the slices were taken was eight and eight, for the knockout and controls, respectively.

Figure 4

Paired-pulse facilitation is normal in hippocampal slices from conditional neuronal NT-3 mutant mice. Shown is the mean for three different interstimulus intervals. There was no significant difference between the knockout (solid bars) and control (open bars) slices at any of the intervals tested. The number of slices used for each interval is as follows (knockout/control): 10 msec (5,5); 25 msec (8,8); 50 msec (20,21); 100 msec (20,21). The number of animals from which the slices were taken was 8 and 8 for the 50 and 100 msec ISI, 3 and 3 for the 25 msec ISI, and 2 and 2 for 10 msec ISI for the knockout and controls, respectively.

Figure 5

LTP is normal in hippocampal slices form conditional neuronal NT-3 mutant mice. (a) Ensemble averages for slices exposed to two epochs of 100 Hz stimulation (tetanus) for 1 sec each. Both the control and the mutant mice exhibited LTP, which was of a similar magnitude. In the mutant mice the mean fEPSP slope was −0.25 ± 0.02 mV/msec and −0.38 ± 0.02 mV/msec, before and after LTP induction by 100 Hz. These slices exhibited significant potentiation (P < 0.001). In the control mice the mean fEPSP slope was −0.24 ± 0.01 mV/msec and −0.41 ± 0.02 mV/msec, before and after LTP induction by 100 Hz. These slices also exhibited significant potentiation (P < 0.001). (n) Number of slices. The number of animals from which the slices were obtained was four and four for knockout and control mice, respectively. (b) Ensemble averages for slices exposed to TBS. Both the control and the mutant mice exhibited LTP, which was of a similar magnitude. In the mutant mice the mean fEPSP slope was −0.25 ± 0.02 mV/msec and −0.36 ± 0.03 mV/msec, before and after LTP induction by TBS. These slices exhibited significant potentiation (P < 0.001). In the control mice the mean fEPSP slope was −0.23 ± 0.02 mV/msec and −0.32 ± 0.03 mV/msec, before and after LTP induction by TBS. These slices also exhibited significant potentiation (P < 0.001). Bar, 0.5 mV/100 msec. (n) Number of slices. The number of animals from which the slices were obtained was four and four for knockout and control mice, respectively.