Regional and cellular expression of netrin-G1 and netrin-G2 mRNAs in human temporal cortex
Using pan probes specific for each gene, NTNG1 and NTNG2 mRNAs were detected in the human temporal cortex. Minimal background signal was seen for sections incubated in the presence of excess cold unlabelled probe, or after hybridization with probes in the sense direction (data not shown).
Regionally, no NTNG1 mRNA signal was detected in the hippocampus proper, with low to moderate signal observed over the subiculum, parahippocampal gyrus, and perirhinal and inferior temporal cortex (). Examination of dipped sections revealed that NTNG1 mRNA was expressed in occasional hippocampal interneurons (), and some subicular pyramidal neurons (). In the perirhinal and inferior temporal cortex, NTNG1 mRNA was mostly localized over layer 3 neurons ().
Figure 2 Regional distribution of (A) NTNG1 and (B) NTNG2 mRNAs in the temporal lobe of representative control subjects. CA: cornu ammonis; DG: dentate gyrus; ITC: inferior temporal cortex; SUB: subiculum: PHG: parahippocampal gyrus; PRC: perirhinal cortex. Although (more ...)
Figure 3 Cellular expression of (A-C, E) NTNG1 and (D, F) NTNG2 mRNAs in (A-D) human temporal lobe and (E, F) P42 rat hippocampal formation. A: NTNG1 mRNA is expressed by occasional putative hippocampal interneurons (arrow), as observed here in the stratum oriens (more ...)
NTNG2 mRNA was abundant in DG, CA4 and CA3, and the deep layers of the perirhinal and inferior temporal cortex. Signal was moderate in CA1, minimal in the subiculum, and variable in the parahippocampal cortex (). At the cellular level, NTNG2 mRNA was concentrated over DG granule cells and pyramidal neurons in each subfield, but was not convincingly observed over putative interneurons (data not shown). In the perirhinal and inferior temporal cortex, NTNG2 mRNA was strongly expressed by neurons in deep layer 5 and layer 6 ().
In summary, both NTNG1 and NTNG2 mRNAs are predominantly expressed by excitatory neurons in the human temporal cortex, but are concentrated in different cell populations and areas of the temporal lobe, and with NTNG1 mRNA also being expressed by some putative inhibitory neurons. Overall, NTNG2 mRNA is more abundantly expressed than NTNG1 mRNA.
Regional and cellular expression of netrin-G1 and netrin-G2 mRNAs in rat hippocampal formation
As in the human, in the rat hippocampus NTNG2 mRNA was more abundant than NTNG1 mRNA, though some species differences in the distribution of NTNG1 and NTNG2 mRNAs between rats and humans were found. For example, in contrast to humans, NTNG1 mRNA was expressed at relatively high levels in the adult rat DG (Figures and ). In addition, at the cellular level in the adult rat, NTNG1 mRNA was localized over the pyramidal layer of CA1 and granule cells of the DG, and was also expressed by hippocampal interneurons (). Expression of NTNG1 mRNA in CA1 was especially prominent during early hippocampal development ().
Figure 4 Representative autoradiographic images demonstrating the regional distribution of (A-D) NTNG1 and (E-J) NTNG2 mRNAs in the developing rat hippocampus. Arrow in E indicates the location of the developing hippocampus. NTNG1 mRNA was not detected in the (more ...)
As observed in humans, NTNG2 mRNA was more abundantly expressed than NTNG1 mRNA, though it was expressed at relatively high levels throughout most hippocampal subfields (), with NTNG2 mRNA concentrated over granule cells of the DG () and pyramidal neurons in CA3 and CA1, but not convincingly over putative interneurons ().
NTNG1 isoform expression during human brain development
Developmental changes in NTNG1 isoform expression were examined in humans using total RNA from fetal brain and adult hippocampus and temporal cortex. Semi-quantitative RT-PCR detected several PCR products of the predicted sizes. In fetal brain and adult temporal cortex, these corresponded to G1c, G1d, G1e and G1m whilst in adult hippocampus, an additional band corresponding to G1a was detected (data not shown). The four NTNG1 isoforms that were detected in all samples examined (G1c, G1d, G1e, G1m), expressed of a percentage of their sum abundance, is shown in . Whilst G1c, G1d, G1e and G1m are all detected from early brain development onwards, the relative expression of each isoform changes in adulthood (). Of note, the relative abundance of G1c and G1d shifts with development, with G1c becoming the predominant isoform in adult hippocampus and temporal cortex.
Figure 5 Relative expression of NTNG1 isoform mRNAs during human brain development. Four isoforms (G1c, G1d, G1e, and G1m) were reliably detected in fetal brain and adult hippocampus and temporal cortex. The relative abundance (expressed as a percentage of the (more ...)
Developmental changes in netrin-G1 and netrin-G2 mRNAs in rat hippocampus
Developmental shifts in expression of NTNG1 and NTNG2 mRNAs were seen in the rat hippocampus (), with a profile that differed between subfields and transcripts. NTNG1 mRNA was first detected reliably at P3. In the DG, NTNG1 mRNA was weakly expressed until P14, with significant increases between P14 and P22 (P=0.046), and between P22 and P42 (P<0.001). In CA1, NTNG1 mRNA decreased between P7 and P14 (P=0.005). For NTNG2, mRNA signal decreased steadily in CA1 from E19 through to P14, but increased in DG between E19 and P3 (P=0.016), and again between P22 and P42 (P=0.037). In CA3, NTNG2 mRNA increased between P3 and P7 (P=0.001), after which it decreased, reaching significance between P14 and P22 (P=0.002).
Summary of developmental changes in NTNG1 and NTNG2 mRNAs during rat hippocampal development.
Netrin-G1 mRNA isoform expression in schizophrenia and bipolar disorder
Using the same semi-quantitative RT-PCR method as utilized in the study of NTNG1 isoform expression during human brain development described above, five NTNG1 PCR products (G1a, G1c, G1d, G1e and G1m) were readily detectable in adult human temporal lobe from the Stanley Medical Research Institute array collection ()
Figure 6 Examples of gel-like images produced by the Agilent 2100 bioanalyzer for semi-quantitative analysis of NTNG1 mRNA using RT-PCR, showing a representative control subject (C), a subject with schizophrenia (S), and a bipolar disorder subject (BP). Bands (more ...)
Two-way ANCOVA (with age, post mortem delay and RIN as covariates) demonstrated a significant effect of diagnosis (F2, 497 = 3.88, P=0.021) and isoform (F4, 497 = 276.59, P<0.001), and a significant diagnosis-by-isoform interaction (F8, 497 = 2.25, P=0.023), on NTNG1 mRNA. Subsequent one-way ANCOVAs revealed a significant effect of diagnosis upon G1c mRNA, but not for any other isoform (). As shown in , G1c mRNA was significantly decreased in bipolar disorder subjects as compared to controls, with a similar trend in schizophrenia that became significant (P=0.037) if the analysis was repeated with the omission of the one outlier (defined as > 2 SDs from the group mean). We also quantified G1c mRNA in a subset of subjects using qPCR to assess the quantitative nature of our RT-PCR method. A significant correlation was detected between G1c mRNA measures using cDNA obtained from the same reverse transcription reaction for each subject (r=0.844, P<0.001). These findings, together with the absence of any change in the expression of the other NTNG1 isoforms examined, indicate that decreased G1c mRNA is unlikely to be due to any chance difference between the groups in the amount of starting mRNA used.
NTNG1 isoform expression in the temporal cortex in controls and subjects with schizophrenia or bipolar disorder
Figure 7 Quantitative analyses of NTNG1 and NTNG2 mRNA expression in the temporal lobe in controls (squares), subjects with schizophrenia (triangles), and subjects with bipolar disorder (circles). A: G1c isoform of NTNG1 mRNA in homogenized temporal cortex tissue. (more ...)
The abundance of all NTNG1 isoforms correlated with RIN (R≥0.245, P<0.013), and all except G1e also correlated with brain pH (R≥0.294, P<0.003), but there were no correlations with age nor post mortem delay (R≤−0.138, P≥0.165). As described in the Methods, for our secondary analyses we therefore repeated the ANCOVAs using only RIN, and pH for all isoforms except G1e, as the covariates. This retained the significant diagnosis-by-isoform interaction, but the reduced G1c mRNA expression in bipolar disorder became non-significant (data not shown). NTNG1 isoform expression did not correlate with lifetime antipsychotic drug exposure nor total inpatient time in either subjects with schizophrenia or bipolar disorder, except for G1e mRNA expression in schizophrenia, which correlated significantly with lifetime drug exposure (r=−0.453, P=0.007). Alcohol, substance misuse and smoking had no significant effects upon NTNG1 isoform expression (data not shown).
Netrin-G2 mRNA expression in schizophrenia and bipolar disorder
Two-way ANCOVA (with age, post mortem delay and RIN as covariates) detected significant effects of diagnosis (F2, 494 = 3.707, P=0.025) and subfield (F4, 494 = 191.27, P<0.001) upon NTNG2 mRNA, but with no diagnosis-by-subfield interaction (F8, 494 = 0.57, P=0.801). Nevertheless, one-way ANCOVAs were performed for each subfield, and showed that diagnosis had a significant effect on NTNG2 mRNA in CA4 and CA3, with a trend in the perirhinal cortex, and no changes in DG or inferior temporal cortex (). In CA4 () and perirhinal cortex (), NTNG2 mRNA was reduced in bipolar disorder as compared to control subjects, whilst in CA3 (), NTNG2 mRNA was decreased in subjects with bipolar disorder and schizophrenia.
Significant inverse correlations between age and NTNG2 mRNA were detected in CA4 (R=−0.234, P=0.017), CA3 (R=−0.209, P=0.033) and the inferior temporal gyrus (R=−0.306, P=0.002). Brain pH correlated with NTNG2 mRNA in most subfields (R≥0.216, P≤0.033) except DG (R=0.129, P=0.192), whilst RIN correlated with NTNG2 mRNA signal in all subfields (R≥0.202, P≤0.047). When the ANCOVAs were repeated including only the significantly correlated variables as covariates, the significant effects of diagnosis seen in CA4, CA3 and perirhinal cortex remained, and no additional significant effects emerged.
Lifetime antipsychotic exposure correlated inversely with NTNG2 mRNA in DG (r=−0.336, P=0.049), CA4 (r=−0.448, P=0.007), CA3 (r=−0.382, P=0.024) and inferior temporal cortex (r=−0.38, P=0.032) in schizophrenia. Total inpatient time did not correlate with NTNG2 mRNA. Neither smoking nor alcohol had any significant effects upon NTNG2 mRNA in any subfield. Substance misuse affected NTNG2 mRNA in CA4 (F5, 61=4.209, P=0.002) and CA3 (F5, 61=4.476, P=0.002), with expression being lower in subjects with no history of misuse.
Schizophrenia associated SNPs and netrin-G1 and netrin-G2 mRNA expression
Genotypes for the two SNPs examined, rs1373336 and rs1105684, were in Hardy-Weinberg equilibrium in each diagnostic group. NTNG1 and NTNG2 mRNAs were not influenced by the respective disease associated SNPs examined, and no genotype-by-diagnosis interactions were detected ().
NTNG1 and NTNG2 genotype is not associated with mRNA expression.
Effects of antipsychotic drug treatment on netrin-G1 and netrin-G2 mRNAs in rat hippocampus
Two-way ANOVA showed that 2 weeks' antipsychotic drug treatment did not affect NTNG1 mRNA expression (F5, 42 =0.521, P=0.598), but did influence NTNG2 mRNA (F8, 63 = 4.49, P=0.015). Subsequent one-way ANOVAs demonstrated that NTNG2 mRNA was unaffected by haloperidol, whilst small but significant increases in NTNG2 mRNA were detected in the DG and CA1 with clozapine ().
Effect of antipsychotic drug treatment on NTNG1 and NTNG2 mRNAs in rat brain.