A possible mechanism for improvement by a cognition-enhancer nefiracetam of spatial memory function and cAMP-mediated signal transduction system in sustained cerebral ischaemia in rats

Abstract

1. Accumulated evidence indicates that the adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signal transduction system may be linked to learning and memory function. 2. The effects of nefiracetam, which has been developed as a cognition enhancer, on spatial memory function and the AC/cAMP/PKA/CREB signal transduction system in rats with sustained cerebral ischaemia were examined. 3. Microsphere embolism (ME)-induced sustained cerebral ischaemia was produced by injection of 700 microspheres (48 micro m in diameter) into the right hemisphere of rats. Daily oral administration of nefiracetam (10 mg kg(-1) day(-1)) was started from 15 h after the operation. 4. The delayed treatment with nefiracetam attenuated the ME-induced prolongation of the escape latency in the water maze task that was examined on day 7 to 9 after ME, but it did not reduce the infarct size. 5. ME decreased Ca(2+)/calmodulin (CaM)-stimulated AC (AC-I) activity, cAMP content, cytosolic PKA Cbeta level, nuclear PKA Calpha and Cbeta levels, and reduced the phosphorylation and DNA-binding activity of CREB in the nucleus in the right parietal cortex and hippocampus on day 3 after ME. The ME-induced changes in these variables did not occur by the delayed treatment with nefiracetam. 6. These results suggest that nefiracetam preserved cognitive function, or prevented cognitive dysfunction, after sustained cerebral ischaemia and that the effect is, in part, attributable to the prevention of the ischaemia-induced impairment of the AC/cAMP/PKA/CREB signal transduction pathway.

Figure 1

The time course of changes in neurological deficits of nefiracetam-untreated (closed circles) and nefiracetam-treated microsphere-embolized rats (open circles). The neurological deficits were scored on the basis of paucity of movement, truncal curvature, and forced circling during locomotion. The rats that had a total score of 7–9 points on day 1 after the operation were used in the present study. No neurological deficits of the nefiracetam-untreated and nefiracetam-treated sham-operated rats were observed. Each value represents the mean±s.e.mean (n=10 each).

Figure 2

Changes in the escape latency in the water maze task of the nefiracetam-untreated (closed circles) and nefiracetam-treated (open circles) rats with ME, and nefiracetam-untreated sham-operated rats (open diamonds). Changes in the escape latency of the nefiracetam-treated sham-operated animals were quite similar to those of the corresponding untreated animals, as described in Table 1. Thus, the former changes are not shown in this figure. The escape latency was determined on day 7 to 9 after the ME. Each value represents the mean±s.e.mean of 10 animals. Two-way ANOVA of the data revealed significant differences in the escape latency between the sham-operated and ME rats (P<0.0001), and between nefiracetam-untreated and nefiracetam-treated rats with ME (P<0.0001). ^*Significantly different (P<0.05) from the sham-operated group when estimated by Fisher's post-hoc PLSD. ^†Significantly different (P<0.05) from the corresponding untreated ME group when estimated by Fisher's PLSD. #Significantly different (P<0.05) from the sham-operated group when estimated by Fisher's PLSD.

Figure 3

TTC-staining of the brain slices at 2, 4, 6, 8, 10, 12, and 14 mm from the forebrain of the microsphere-embolized and nefiracetam-treated microsphere embolized rats on day 10. There was no significant difference in the infarct areas between the two groups (P=0.636).

Figure 4

Representative Western immunoblots of GTP-binding protein subunits (Gα_s, Gαα_i1,2, and Gβ) indicating the specific 52, 45-kDa band for Gα_s, 40-kDa one for Gα_i1,2, and 35-kDa one for Gβ of the right parietal cortex (A) and hippocampus (B) from the right hemisphere of the nefiracetam-untreated (ME) and nefiracetam-treated (MN) rats with ME, and from that of the nefiracetam-untreated (S) and nefiracetam-treated (SN) sham-operated rats on day 3 after the operation. There were no differences in Gα_s, Gα_i1,2, and Gβ protein levels among these groups.

Figure 5

Representative Western immunoblots of AC-I, AC-V/VI, and AC-VIII in the parietal cortex (A) and hippocampus (B) from the right hemisphere of the nefiracetam-untreated (ME) and nefiracetam-treated (MN) rats with ME, and from that of the nefiracetam-untreated (S) and nefiracetam-treated sham-operated (SN) rats on day 3 after the operation (upper panel). Quantified data for the levels of AC-I protein in the right parietal cortex and hippocampus are shown in the lower panels. Each value represents the mean percentage of the control±s.e.mean of eight animals. ^*Significantly different from the sham-operated group (P<0.05). ^†Significantly different from the corresponding untreated ME group (P<0.05). #Significantly different from the nefiracetam-treated sham-operated (SN) group (P<0.05).

Figure 6

cAMP content of the parietal cortex (A) and hippocampus (B) in the right hemisphere of the nefiracetam-untreated (ME) and nefiracetam-treated (MN) rats with ME, and of the nefiracetam-untreated (S) and nefiracetam-treated (SN) sham-operated rats on day 3 after the operation. Each value represents the mean±s.e.mean of six (S and SN) or eight (ME and MN) animals. ^*Significantly different from the sham-operated group (P<0.05). ^†Significantly different from the corresponding untreated ME group (P<0.05). #Significantly different from the nefiracetam-treated sham-operated (SN) group (P<0.05).

Figure 7

Representative Western immunoblots of nuclear PKA subunits (Cα and Cβ) in the upper panels indicate the specific 42-kDa band for Cα in the right parietal cortex (A) and hippocampus (B), and the 55-kDa band for Cβ in the parietal cortex (C) and hippocampus (D) from the right hemisphere of the nefiracetam-untreated (ME) and nefiracetam-treated (MN) rats with ME, and from that of the nefiracetam-untreated (S) and nefiracetam-treated (SN) sham-operated rats on day 3 after the operation. Quantified data for the levels of Cα and Cβ proteins in the right parietal cortex and hippocampus are shown in the lower panels. Each value represents the mean percentage of the control±s.e.mean of eight animals. ^*Significantly different from the sham-operated group (P<0.05). ^†Significantly different from the corresponding untreated ME group (P<0.05). #Significantly different from the nefiracetam-treated sham-operated (SN) group (P<0.05).

Figure 8

Representative Western immunoblots of pCREB and total CREB in the upper panels indicate the specific 43-kDa band for pCREB in the parietal cortex (A) and hippocampus (B), and total CREB of the parietal cortex (C) and hippocampus (D) in the right hemisphere of the nefiracetam-untreated (ME) and nefiracetam-treated (MN) rats with ME and in that of the nefiracetam-untreated (S) and nefiracetam-treated (SN) sham-operated rats on day 3 after the operation. Quantified data for the levels of pCREB protein and total CREB protein of the right parietal cortex and hippocampus are shown in the lower panels. Each value represents the mean percentage of the control±s.e.mean of eight animals. ^*Significantly different from the sham-operated group (P<0.05). ^†Significantly different from the corresponding untreated ME group (P<0.05). #Significantly different from the nefiracetam-treated sham-operated (SN) group (P<0.05).

Figure 9

EMSA of CREB-binding activity in the right parietal cortex and hippocampus. Representative autoradiograms on the left show CREB-binding in the right parietal cortex (A) and hippocampus (D). The first lanes in Figure 9A,D (control) show a retardation caused by CREB binding to the CRE oligonucleotide in the absence of an antibody or competitor oligonucleotides. The addition of CREB antibody supershifted entirely the shifted band (the second lanes), as marked by arrowheads. In addition, the presence of excess cold CRE oligonucleotide blocked the binding of the CREB protein to the radioactive CREB band (the third lanes), whereas excess AP-1 oligonucleotide had no effect on the CREB binding (the fourth lanes), as marked by arrows. The finding indicates that the shift was caused by binding of CREB-related proteins. Representative autoradiograms in the middle indicate the specific band for CREB-CRE binding in samples from the right parietal cortex (B) and hippocampus (E) from the right hemisphere of the nefiracetam-untreated (ME) and nefiracetam-treated (MN) ME rats, and from that of the nefiracetam-untreated (S) and nefiracetam-treated (SN) sham-operated rats on day 3 after the operation. Quantified data for the level of CREB binding activity of the right parietal cortex (C) and hippocampus (F) are shown at the right. Each value represents the mean percentage of the control±s.e.mean of eight animals. ^*Significantly different from the sham-operated group (P<0.05). ^†Significantly different from the corresponding untreated ME group (P<0.05).