• Alzheimer’s disease;
  • β-amyloid;
  • cortical neurons;
  • neurotoxicity;
  • somatostatin


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In β-amyloid (Aβ)-induced neurotoxicity, activation of the NMDA receptor, increased Ca2+ and oxidative stress are intimately associated with neuronal cell death as normally seen in NMDA-induced neurotoxicity. We have recently shown selective sparing of somatostatin (SST)-positive neurons and increased SST expression in NMDA agonist-induced neurotoxicity. Accordingly, the present study was undertaken to determine the effect of Aβ25–35-induced neurotoxicity on the expression of SST in cultured cortical neurons. Cultured cortical cells were exposed to Aβ25–35 and processed to determine the cellular content and release of SST into medium by radioimmunoassay and SST mRNA by RT-PCR. Aβ25–35 induces neuronal cell death in a concentration- and time-dependent fashion, increases SST mRNA synthesis and induces an augmentation in the cellular content of SST. No significant changes were seen on SST release at any concentration of Aβ25–35 after 24 h of treatment. However, Aβ25–35 induces a significant increase of SST release into medium only after 12 h in comparison with other time points. Most significantly, SST-positive neurons are selectively spared in the presence of a lower concentration of Aβ25–35, whereas, in the presence of higher concentrations of Aβ25–35 for extended time periods, SST-positive neurons decrease gradually. Furthermore, Aβ25–35 induces apoptosis at lower concentrations (5 and 10 μmol/L) and necrosis at higher concentrations (20 and 40 μmol/L). Consistent with the increased accumulation of SST, these data suggest that Aβ25–35 impairs cell membrane permeability. Selective sparing of SST-positive neurons at lower concentrations of Aβ25–35 at early time points directly correlates with the pathophysiology of Alzheimer’s disease.

Abbreviations used



Alzheimer’s disease


brain-derived neurotrophic factor


Dulbecco’s modified Eagle’s medium


fetal bovine serum


glyceraldehyde-3-phosphate dehydrogenase


lactate dehydrogenase


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


neuron-specific enolase


Huntington’s disease


horse serum


NMDA receptor


phosphate-buffered saline


quinolinic acid




SST-like immunoreactivity


Tris-buffered saline

Somatostatin (SST), a multifunctional peptide, was first described as a neurotransmitter and neuromodulator (Reichlin 1983). Increasing evidence suggests that disturbances of the somatostatinergic system are involved in several human neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease, Huntington’s disease (HD), hypoxic–ischemic neuronal injury, neoplasia, prion disease and acquired immune-deficient syndrome encephalopathy (Agren and Lundqvist 1984; Beal et al. 1985; Vecsei et al. 1990; Banki et al. 1992; Krantic et al. 1992). In AD brain tissue, the elimination of SST in the cortex and hippocampus is directly linked to poor cognitive function and memory impairment (Davies et al. 1980; Grouselle et al. 1998). Furthermore, reduced concentration of SST in AD is frequently observed in cortical and cerebrospinal fluid (Davies et al. 1980; Beal et al. 1988; Bisette et al. 1998; Davis et al. 1999; Nemeroff 1999). In addition, SST also decreases in a age-dependent manner (Hayashi et al. 1997; Lu et al. 2004). Unlike in AD, SST levels in HD are increased in striatal and basal ganglia and intimately associated with motor coordination disability in Huntington’s chorea (Beal et al. 1986). Also subsets of SST-producing neurons are selectively resistant in NMDA agonist-induced neurodegeneration (Dawbarn et al. 1985; Ferrante et al. 1987; Kumar 2004). In parallel, it has also been shown that SST exerts a neuroprotective effect against NMDA-mediated toxicity (Forloni et al. 1997).

Previous studies have shown that β-amyloid (Aβ) increases the vulnerability of cultured cortical neurons to excitotoxic damage through glutamatinergic pathways. The increased accumulation of Aβ42, a cleavage product of β-amyloid precursor protein that accumulates into fibrils in AD brains, is neurotoxic and contributes to dementia (Yankner et al. 1990). Most in vitro data suggest that the amyloid fibril is the neurotoxic form of Aβ, whereas soluble Aβ and non-fibrillar aggregates are non-toxic (Lorenzo and Yankner 1994). In fact, it has been shown that, in culture, the Aβ protein is neurotrophic to undifferentiated hippocampal neurons at lower concentrations but is neurotoxic to mature neurons at higher concentrations (Yankner et al. 1990). Several previous studies investigating AD pathology have directly shown that SST-like immunoreactivity (SSTLI) is reduced in the cerebral cortex and cerebrospinal fluid as well as decreased in SST-binding sites in the frontal cortex in AD cases (Davies et al. 1980; Wood et al. 1982; Beal et al. 1985). Consistent with these data, we have recently shown the loss of SST-immunopositive neurons as well as somatostatin receptor-specific changes in AD cortex in comparison with age-matched normal brains (Kumar 2005). In AD cases, a lower level of SST mRNA expression, per somatostatinergic cell in the hippocampus, has been shown (Dournaud et al. 1994). However, it is currently unknown whether the changes in SST seen in in vivo can also be attributed in Aβ-induced neurotoxicity.

In an attempt to elucidate the role of the somatostatinergic system in neurodegenerative diseases such as AD, the mechanism of glutamate toxicity was initially investigated. Additionally, quinolinic acid (QUIN), NMDA receptor (NMDAR) agonist, mimicked a model of HD and appeared to increase SST gene expression and peptide production (Patel et al. 1991). In Aβ-induced neurotoxicity, NMDAR activation and increase in Ca2+ are frequently seen. This is similar to what has been reported for NMDA-induced excitotoxicity. High levels of Aβ deposit alongside decreased levels of SST in AD brain tissue further incited us to elucidate the possible role of SST in Aβ-induced neurotoxicity. Our preliminary data support the concept that Aβ influences SST release and its accumulation in cortical neurons (Geci et al. 2000). Recently, Sato et al. (2004) showed that SST regulates the metabolism of Aβ through modulatory proteolytic degradation catalyzed by neprilysin. Accordingly, in the present study, employing cortical cell culture as an in vitro model, we tested the hypothesis that Aβ25–35 induces SST gene transcription, protein production, and secretion. Our data clearly demonstrate an increase in SST accumulation in cortical neurons and release of SST into culture medium in a time- and concentration-dependent manner in response to Aβ25–35-induced neurotoxicity.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References


Dulbecco’s modified Eagle’s medium (DMEM), horse serum (HS) and fetal bovine serum (FBS) were obtained from Wisent (Montreal, Quebec, Canada), the antibiotic solution of streptomycin/penicillin was from Gibco BRL (Carlsbad, CA, USA), and 5-fluor-2′-deoxyuridine and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemicals (Oakville, ON, Canada). Aβ25–35 was purchased from Bachem (Torrance, CA, USA).

Primary cortical cultures

Primary culture of cortical neurons was prepared from 15- to 16-day-old rat embryonic brains essentially as described earlier (Kumar et al. 1997; Kumar 2004). Briefly, embryos were collected in DMEM containing 10% HS, 2% FBS, 2 mmol/L glutamine and 1% penicillin and streptomycin. Cortices from 10 to 14 embryos were isolated and incubated at 37°C for 20 min in 0.027% trypsin in pre-warmed Brooks–Logan solution [5% phosphate-buffered saline (PBS), 40 mmol/L sucrose, 30 mmol/L glucose, 10 mmol/L HEPES, pH 7.4] with gentle swirling. The supernatant was carefully decanted and the trypsinized tissue mass was triturated in 10 mL of DMEM containing 10% HS, 2% FBS, 2 mmol/L glutamine, penicillin and streptomycin. The cells were seeded on poly-l-ornithine-coated glass coverslips in 24-well plates at a density of 3–4 × 106 cells/well and maintained undisturbed for 4 days at 37°C with 5% CO2 in a humidified incubator. On the fifth day, the culture medium was aspirated and the cells were incubated for four additional days in DMEM containing 10 μg/mL 5-fluoro-2-deoxyuridine to inhibit glial cell proliferation. Neuronal cells were subsequently maintained in DMEM containing 5% HS, 2 mmol/L glutamine, penicillin and streptomycin with a change of media twice weekly. Studies were carried out in 10- to 12-day-old neuronal cultures.


To study the expression of SST and neuron-specific enolase (NSE), immunoreactivity neurons were fixed in 4% paraformaldehyde for 20 min, washed three times with Tris-buffered saline (TBS) and incubated with 5% normal goat serum in TBS with 0.2% Triton X-100 for 1 h. Immunocytochemistry was performed as described earlier (Kumar et al. 1997; Kumar 2004). Briefly, neurons were incubated with rabbit polyclonal anti-SST (1 : 1000) and monoclonal NSE antibodies (1 : 1500) overnight. Following three subsequent washes with TBS, the cells were incubated with a goat anti-rabbit or goat anti-mouse Cy3-labeled secondary antibody (1 : 400) in TBS for 1 h. After three subsequent washes in TBS, coverslips were mounted in immunofluor mounting medium and then viewed and photographed by using a Leica fluorescent microscope (Wetzlar, Postfach, Germany).


To examine the effect of Aβ25–35 on cultured cortical neurons, cultures were washed three times in control salt solution (containing 120 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2, 25 mmol/L Tris–HCl, and 15 mmol/L glucose, pH 7.4) and exposed to increasing concentrations of Aβ25–35 (5–40 μmol/L) in serum-free DMEM for 24 h at 37°C. The medium was subsequently removed and replaced by standard medium and maintained for an additional 24 h. Cultures were washed with PBS and exposed to trypan blue (0.4% in PBS) for 10–15 min. Cells stained with trypan blue (non-viable cells) and non-stained cells (viable cells) were counted from 10 different randomly selected areas from each culture and calculated as percentage of control (without Aβ). The results of these cytotoxicity assays were also confirmed by cresyl violet staining which revealed comparable data. In order to assess the time-dependent effects of Aβ, cultures were incubated for different time periods (12–72 h) at 37°C and data were analyzed as described above to quantitate the survival of neurons.

MTT reduction assay

In addition to conventional trypan blue exclusion assay, quantification of neuronal viability was further confirmed by MTT assay, which measures the ability of viable cells to reduce MTT. Briefly, the culture medium from control and Aβ25–35-treated cultures was removed and replaced with 1 mL of RPMI-1640 lacking phenol red and containing 1.5 mg/mL MTT. The cells were incubated for 3–4 h at 37°C, after which the MTT solution was removed and the formazan crystals solubilized by incubation in isopropanol with 0.1 n hydrochloric acid for a minimum of 5 h. The absorbance of the lysate was measured between 570 and 690 nm using a spectrophotometer (Pharmacia Biotech, Uppsala, Sweden).

Reverse transcriptase-polymerase chain reaction

A semi-quantitative RT-PCR assay was used to determine the mRNA levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Total RNA from cortical neurons was isolated by TRIZOL extraction, reverse transcribed, and the resulting cDNAs were amplified by PCR using the following specific SST primers:



Somatostatin was quantified from ethidium bromide-stained gels of the PCR reaction with Canon Craft CS-P37 software (Mississauga, Ontario, Canada) and used as an index of SST and GAPDH mRNA. The units derived from this analysis were arbitrarily assigned an optical density corrected for background. Values of mRNA expression were normalized to those of GAPDH mRNA on the same gels. The molecular size for SST is 280 and 240 bp for GAPDH.


To determine SSTLI in neuronal cells, individual culture wells from control and Aβ25–35-treated cells were extracted in 1 n acetic acid from media as previously described (Patel et al. 1991). SSTLI in cells and media was measured by radioimmunoassay using an anti-SST antibody directed against the central segment of SST-14, [125I-Tyr] SST-14 radioligand and SST-14 standards .This assay detects SST-14 and SST-28 equally and recognizes the amino-terminally extended form of SST-28 including pro-SST.

Determination of apoptosis and necrosis in cortical-cultured neurons

To determine the nature of cell death, cortical-cultured neurons were exposed to different concentrations (5–40 μmol/L) of Aβ25–35 for 24 h. Following treatment, cultures were subsequently washed in PBS and fixed in 4% paraformaldehyde for 20 min. Cultures were washed and treated with 0.2% Triton X-100 for 10 min and followed by incubation with Hoechst 33258 at a concentration of 1 µg/mL for 30 min at 37°C to determine apoptotic cells. To determine the number of necrotic cells, cultured cortical neurons were incubated with propidium iodide (50 µg/mL) in PBS for 15 min at 20°C. Cells were washed in PBS, mounted and photographed using fluorescence microscopy.

Statistical analysis

Data were expressed as mean ± SE. Statistical comparisons were made using one-way anova and Dunnett’s multiple comparison tests.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Time- and concentration-dependent effect of β-amyloid on cortical neurons

In an initial experiment, we compared the effect of Aβ25–35 on morphological changes and neuronal survival of cortical neurons in time- and concentration-dependent manner. Primary embryonic rat cortical cultures were incubated with 20 μmol/L of Aβ25–35 for 12–72 h or in the presence of different concentrations of Aβ25–35 (10–40 μmol/L) for 24 h and processed for cell morphology and cell viability. As shown in Fig. 1(a), in the presence of Aβ25–35 (20 μmol/L), neuronal culture displayed a gradual deterioration in morphology and neuronal loss in a time-dependent manner [Fig. 1a(ii–vi)] when compared with control [Fig. 1a(i)]. In Aβ25–35-induced neurotoxicity, prominent features included degeneration of neuronal processes followed by membrane blabbing and swelling of neuronal perikarya. Following 48- and 72-h treatment with Aβ25–35, the majority of neurons were lost [Fig. 1a(v,vi)].


Figure 1.  Time- and concentration-dependent effect of Aβ25–35 in cortical neurons. Cortical neurons were treated for 12–72 h in the presence of 20 μmol/L or 24 h in the presence of different concentrations (10–40 μmol/L) of Aβ25–35. After treatment, culture was processed for morphological changes as well as for cell survival. (a) Aβ (20 μmol/L) caused neuronal damage and degeneration of dendritic arbors in a time-dependent manner (panels ii–vi). It severely damaged neurons at 36 h (panel iv) and followed by complete loss of neurons following 48–72 h (panels v and vi) of the treatment in comparison with 0 h (panel i). After treatment, quantification of neuronal cell death was accomplished by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (b) Aβ25–35-induced neuronal cell death in a time-dependent manner. To examine the concentration dependency, cultured cortical cells were incubated at 37°C for 24 h in the presence of increasing concentration (5–40 μmol/L) of Aβ25–35. (c, d) Aβ25–35 displayed significant morphological changes and gradual decrease in neuronal survival in a concentration-dependent manner. In comparison with 5 and 10 μmol/L of Aβ25–35, significant morphological changes and loss of communicating nerve fibers were observed in the presence of 20 and 40 μmol/L Aβ25–35. All data points represent the mean % ± SE of at least three independent experiments performed in triplicate. *p < 0.05, **p < 0.01 using one-way anova and Dunnett’s multiple comparison tests.

Download figure to PowerPoint

In response to 24-h treatment with different concentrations (5–40 μmol/L) of Aβ25–35, some neurons appeared atrophied, and were severely damaged from their axonal processes at the higher concentration [Fig. 1c(iii,iv)]. As shown in Figs 1(c) and (d), Aβ25–35 induced neuronal cell death in a concentration-dependent fashion with cell viability decreasing from 82.70 ± 3.3% to 42.35 ± 4.3% in the presence of varying concentrations (5–40 μmol/L) of Aβ25–35.

β-Amyloid induces concentration- and time-dependent SST gene expression

In an initial experiment, we compared the effect of different doses of Aβ25–35 on SST mRNA expression. As shown in Fig. 2(a), at the concentrations of 5–20 μmol/L, Aβ25–35 had no significant effect on SST mRNA expression, whereas at a higher concentration (40 μmol/L), Aβ25–35 induced 260% increase in SST mRNA expression within 24 h (Fig. 2a). To ascertain whether the augmentation of SST mRNA by Aβ25–35 was a time-dependent phenomenon, neuronal cultures were exposed to Aβ25–35 (40 μmol/L) for 12–72 h, respectively. As shown in Fig. 2(b), SST mRNA levels were unchanged at 12 h, but increased by 263% at 24 h, and 66% at 48 h. Although SST mRNA decreased by 21% at 72 h in comparison with control (0 h), however, displayed increased mRNA expression when compared with control at 72 h.


Figure 2.  Semi-quantitative analysis of somatostatin mRNA by RT-PCR from control and Aβ25–35-treated cortical-cultured neurons. Total RNA from control and Aβ25–35-treated cells was reverse transcribed as described in Materials and methods. The figures depict representative photographs showing concentration-dependent (a) and time-dependent (b) changes in somatostatin gene expression in response to Aβ25–35. Bottom panels in (a) and (b) represent the densitometric quantitative analysis using GAPDH as control. Data are the mean ± SE of three independent experiments. *p < 0.05.

Download figure to PowerPoint

Concentration- and time-dependent accumulation of SST in response to β-amyloid

Previously, using an NMDAR agonist increased SST gene expression and peptide production has been shown (Patel et al. 1991, 1995). To determine whether similar effects like NMDAR agonists could be observed with Aβ, we next investigated a role for the Aβ25–35 on cellular content of SST. Ten- to 12-day-old cortical cultures were treated with 5–40 μmol/L of Aβ25–35 for 24 h. The concentration of SST in cell extracts was determined by using radioimmunoassay. As shown, addition of Aβ25–35 to cortical neurons up-regulated cellular SST content from basal levels of 6.3 ± 0.4 to 26.4 ± 0.8 ng/mL in a dose-dependent fashion (Fig. 3a). A maximal fourfold increase in SST content was detected in the presence of 40 μmol/L Aβ25–35. We further extended our study and determined time dependency of Aβ25–35 on SST cellular content. Cells incubated with Aβ showed increased cellular SST content by 56% at 12 h, 333% at 24 h, 343% at 48 h and 85% at 72 h, respectively, when compared with control (Fig. 3b). These data strongly suggest that Aβ25–35 induced a concentration- and time-dependent significant enhancement of SST cellular content in cortical neurons.


Figure 3.  Concentration- and time-dependent effect of Aβ25–35 on cellular somatostatin-like immunoreactivity (SSTLI) in cortical neurons. (a) Cultured cortical cells were exposed to increasing concentration (5–40 μmol/L) of Aβ25–35 for 24 h at 37°C. The concentration of SSTLI in cell extracts increased gradually with treatment and peaked at 48 h. (b) Cellular SSTLI level increased in a time-dependent manner. Somatostatin accumulation reached at peak in 48 h and returned to control level after 72 h of incubation. Data are mean ± SE of three independent experiments performed in triplicate. *p < 0.05, **p < 0.01.

Download figure to PowerPoint

Concentration- and time-dependent release of SST in response to β-amyloid

We further extended our study and determined SST release into the culture medium in response to Aβ25–35. Upon incubation of cortical neurons for 24 h in the presence of increasing concentrations of Aβ25–35 (5–40 μmol/L), no significant changes in SST release were observed. As shown in Fig. 4(a), Aβ25–35 stimulated a release of SST from 1.6 ± 0.15 to 2.4 ± 0.09 ng/mL at the concentration of 5–20 μmol/L of Aβ and 2.02 ± 0.11 ng/mL in the presence of 40 μmol/L of Aβ25–35. As SST secretion into culture medium was maintained at control levels upon treatments with different concentrations of Aβ25–35, we next examined the time dependency of SST release in response to Aβ25–35. Cortical-cultured neurons were incubated with 40 μmol/L of Aβ25–35 for 12–72 h. At each time interval, culture medium was collected and processed for SST release. As depicted in Fig. 4(b), SST release was stimulated acutely with a peak response at 12 h. Aβ25–35 evoked a significantly greater SST secretion at this time point and SST increased from 2.98 ± 0.381 to 11.23 ± 1.27 ng/mL (3.8-fold increase), whereas no significant changes were seen in SST release at 24, 48 and 72 h when compared with control values. To see such an acute response from stimulated secretory cells is not surprising. These data are in general agreement with previous studies whereby SST release stimulated two- to fivefold upon treatment with NMDAR agonist, over a 2- to 20-h period, with a fourfold increase at 12 h (Patel et al. 1991). These data suggest that cortical SST is activated in response to Aβ-induced toxicity in both a concentration- and a time-dependent fashion.


Figure 4.  Concentration- and time-dependent release of somatostatin-like immunoreactivity into medium from control or culture incubated with Aβ25–35. To determine the effect of Aβ25–35 on somatostatin release, cortical neurons were incubated in the presence of different concentrations (5–40 μmol/L) of Aβ25–35 for 24 h (a) or with 40 μmol/L of Aβ25–35 for 12–72 h (b) and processed for radioimmunoassay as described in Materials and methods. No significant changes were seen in the presence of any concentration of Aβ25–35 after 24 h of incubation. Somatostatin-like immunoreactivity into medium was significantly greater at 12 h in the presence of 40 μmol/L Aβ25–35. Data are mean ± SE of three independent experiments performed in triplicate. *p < 0.05.

Download figure to PowerPoint

SST immunoreactive neurons are selectively spared in β-amyloid-induced neurotoxicity

We have recently shown that neurons expressing SSTLI are resistant to QUIN- and NMDA-induced neurotoxicity (Kumar 2004). Accordingly, we next determined whether SST-positive neurons are selectively spared or susceptible to Aβ-induced neurotoxicity. Upon treatment with Aβ25–35, there is a concentration- and time-dependent increase in neuronal cell death. NSE-positive neurons were used as an index for total cell count. Figure 5 compares the total neuronal population (NSE-positive neurons) and SST immunoreactive neurons in control or culture treated with 5–40 μmol/L Aβ25–35 for 24 h. In response to Aβ25–35, there was a gradual decrease in NSE-positive neurons; however, SST-positive neurons were selectively spared following 24-h treatment with Aβ25–35. Quantitative analysis of NSE-positive neurons as well as SST-positive neurons in cultures treated with different concentrations of Aβ25–35 (5–40 μmol/L) is shown in Fig. 5 (bottom panels).


Figure 5.  Representative photographs depicting selective sparing of somatostatin (SST)-positive neurons in Aβ25–35-induced neurotoxicity. Cortical neurons were exposed to different concentrations of Aβ25–35 for 24 h. Following treatment, cultures were washed, fixed and incubated in the presence of specific antibodies to neuron-specific enolase (NSE) to determine the total neuronal population in culture. In parallel, cells were incubated with SST-specific antibodies to determine SST-positive cells. The individual neuronal population positive to NSE and SST-like immunoreactivity was photographed from randomly selected areas. In comparison with control culture, there was a gradual decrease in NSE-positive neurons with increasing concentration of Aβ25–35. Note the selective sparing of SST-positive neurons in the presence of Aβ25–35. Quantitative analysis of NSE- and SST-positive neurons is shown in bottom panels, respectively. The number of NSE- and SST-immunopositive neurons was expressed as percentage of total number of neurons positive for NSE in control. Values represent the mean ± SE (n = 9) from three independent experiments performed in triplicate. *p < 0.05.

Download figure to PowerPoint

Characterization of Aβ-induced neurotoxicity in cortical-cultured neurons

As Aβ caused neuronal cell death in a concentration- and time-dependent manner, to ascertain the nature of neuronal cell death, we determined whether Aβ-induced cell death in cortical neurons was apoptotic or necrotic. Cultured neuronal cells were exposed to different concentrations of Aβ25–35 for 24 h and processed for apoptosis (Hoechst 33258 staining) and for necrosis (propidium iodide staining), respectively. In addition, cell morphology was also taken in consideration for apoptosis and necrosis. As shown in Fig. 6(a), incubation of cultured cells at lower concentration of Aβ25–35 (5 and 10 μmol/L) triggered apoptosis within 24 h of treatment. The number of apoptotic cells was 19.8 ± 2% and 12.0 ± 3% at a concentration of 5 and 10 μmol/L, respectively, in comparison with control culture (4%), whereas at higher concentrations Aβ25–35 (20 and 40 μmol/L), the number of apoptotic cells constituted only 5% and 5.6% when compared with control culture. As illustrated in Fig. 6(b), the numbers of necrotic cells gradually increased in a concentration-dependent manner and constituted 10–42% in comparison with control (3.8%). Such increase in necrotic cells is further supported by decrease in MTT assay. These results suggest that at lower concentrations, Aβ25–35 induces apoptosis and this is accompanied by necrosis at higher concentration.


Figure 6.  Representative photomicrographs illustrating apoptosis and necrosis in response to Aβ25–35 in cultured neuronal cells. Two-week-old neuronal cells were incubated for 24 h at 37°C in the presence of increasing concentration of Aβ25–35. Following treatment, cultures were washed, fixed and permeabilized and incubated with Hoechst 33258 1 μg/mL and propidium iodide 50 μg/mL to determine apoptotic and necrotic cells, respectively. The bottom panels in (a) and (b) depict the quantitative analysis of apoptotic and necrotic cells, respectively. Numbers of the cells were counted from 8 to 10 randomly selected areas from each coverslip. Note the increased number of apoptotic cells at lower concentration of Aβ25–35 and gradual increase in necrotic cells with increasing concentration of Aβ25–35. Values represent the mean ± SE (n = 9) from three independent experiments performed in triplicate. *p < 0.05 versus control group.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The results presented here demonstrate an induction of SST mRNA alongside increased SST accumulation and release in cultured cortical neurons exposed to Aβ25–35-induced model of neurotoxicity. The increase in SST mRNA synthesis and SST accumulation is similar to what has been previously reported for NMDA- and QUIN-induced neurotoxicity (Patel et al. 1991). To our knowledge, the present study represents the first description of changes in expression of SST in cultured cortical neurons upon treatment with Aβ25–35. The neuronal culture used in the present study was 98% neuronal after treatment with deoxyuridine for 4 days. Accordingly, the data presented here exclude any possible interference of glial cell. SST expressions and its release in response to other neurotoxins, such as NMDA, have been characterized in depth (Tapia-Arancibia and Astier 1989; Ham et al. 1991, 1993; Patel et al. 1991, 1995; Kumar 2004). Furthermore, this effect of Aβ is not only selective to cultured cortical neurons, but also observed in hypothalamic and striatal cultures (Williams et al. 1991; Rage et al. 1994). The findings pertaining to Aβ-induced SST production recorded in this study are parallel to those described for NMDAR agonist-treated cultures, except for the constant increase in SST release induced by QUIN from 0 to 20 h (Patel et al. 1991).

The number of viable cells decreases significantly when cultures were exposed to synthetic Aβ25–35. In fact, cell viability decreased by 40–60% upon 24-h treatment with 40 μmol/L Aβ as measured by the MTT assay. This level of neuronal death is consistent with several previous studies. It is interesting to note that SST-positive neurons are selectively spared in Aβ25–35-induced toxicity. However, SST-positive neurons become susceptible to Aβ25–35 when exposed to a higher concentration of Aβ25–35 for a more extended period of time. This parallels the pathology of AD, whereby a decrease in SSTLI neurons directly correlates with the severity of the disease (Davies et al. 1980; Wood et al. 1982; Beal et al. 1988; Bisette et al. 1998; Davis et al. 1999; Nemeroff 1999). It should also be noted that the pattern of SST release from cultured cortical neurons treated with increasing concentrations of Aβ25–35 does not mirror the SST accumulation profile occurring simultaneously within the neuron. It would be expected that an increased SST production would translate into higher levels of SST secretion, as SST is primarily a secreted product, acting on neighboring cells as a neurotransmitter or a neuromodulator. Here, although SST production showed fourfold accumulation in cellular content, but the secretion of SST into culture medium remained relatively stable with only a slight enhancement in release was seen after treatment with 20 μmol/L of Aβ25–35. Time course studies of SST induction indicated an acute SST release, four times higher than basal levels, at 12 h with a subsequent return to baseline levels by 24 h. Such an acute response is not uncharacteristic of stimulated secretory cells. Indeed, SST release has been shown to be stimulated two- to fivefold by 10 mmol/L QUIN, over a 2- to 20-h period, with a fourfold increase at 12 h (Patel et al. 1991). Our observations using Aβ25–35 as neurotoxins suggest that Aβ25–35 itself impedes vesicular trafficking inside the cell, consequently affecting SST release. As Aβ25–35 toxicity is believed to be caused in part by the disruption of cholesterol uptake and metabolism, ultimately resulting in abnormal trafficking of membrane proteins which are crucial for normal neuronal function (Lynch and Mobley 2000).

Somatostatin mRNA levels were also affected by Aβ25–35 treatment. It is known that the SST gene is transcriptionally regulated by cAMP-response element binding protein, the cAMP-dependent transcription factor (Gonzalez et al. 1989), but whether these effects are mediated directly or indirectly on SST cells, and whether involve cAMP-response element binding protein in Aβ-mediated neurotoxicity remain to be elucidated. As mentioned previously, steroid hormones, cytokines, growth hormone, and insulin may also affect SST mRNA expression, either directly or indirectly. In addition, it is interesting to note that changes in SST expression have not only been observed in the context of NMDAR or Aβ-mediated neurotoxicity, but also in models of HIV encephalitis (Koutkia et al. 2004). For example, although brain-derived neurotrophic factor (BDNF), an endogenous growth factor, leads to a significant increase in SST production, HIV-1 envelope glycoprotein gp120 and BDNF together lead to an even greater SST production in comparison with that of BDNF alone, in cultured human fetal cortex (Barnea et al. 1999). Therefore, it is possible that SST induction is potentiated by a neurotoxin such as NMDA or Aβ25–35 acting alone or by the synergistic action of an endogenous peptide and a neurotoxin. This implies that the magnitude of SST production and secretion might be under the direct influence of other neuropeptides through different mechanism depending on cell type and neurotoxins.

Although it is not known whether SST involves in apoptosis or not but the role of somatostatin receptor subtypes have been shown in apoptosis. Here, we present the data that lower concentration of Aβ25–35-induced apoptosis and followed by necrosis at the higher concentration by using Hoechst 33258 and PI staining to discriminate apoptotic and necrotic cells, respectively. It has previously been questioned that PI does not discriminate between apoptotic and necrotic cells. In our experimental conditions, we determined necrotic cells on the basis of cell morphology in addition to PI staining. Neuronal cell showing swollen cell bodies and strongly stained with PI was considered apoptotic. Secondly, there was no prominent staining of PI at lower concentration of Aβ25–35, whereas in contrast apoptotic cells were stained strongly with Hoechst 33258 dye but not with PI. Third, lactate dehydrogenase (LDH) release to the culture medium is an index of necrosis, which does not occur in the case of apoptosis. We did not observe any change in LDH release when apoptosis was prominent at lower concentration of Aβ25–35, whereas in the presence of higher concentration of Aβ25–35, LDH was released significantly as an index of dying cells (data not shown). LDH release is supplemented with MTT assay (Fig. 1) for the quantifications of neurons.

Previously, Yankner et al. (1990) demonstrated that Aβ is neurotrophic and that once aggregated, it exerts a neurotoxic effect. However, most significantly, a recent study shows that SST blocks the aggregation of Aβ through the regulation of neprilysin activity (Sato et al. 2004). Furthermore, SST also affects neprilysin expression and its synaptic localization (Sato et al. 2004). These data support our observations with regard to SST release at early time point in response to Aβ and confirm the fact that SST may have an ability to inhibit the aggregation of Aβ. Indeed, we have demonstrated that there is a significant increase in SST release in response to Aβ at 12 h. This increased release of SST could underlie the neurotrophic ability of Aβ by blocking the aggregation of Aβ. However, Aβ may be neurotoxic in the presence of higher concentration when SST release to the culture medium is impaired. As the biological effect of SST is mediated by five different receptor subtypes, it will be essential to determine the roles of individual receptors in Aβ-induced neurotoxicity. Further studies are progressing in this direction. Consistent with gradual reduction of SST in AD brain and aging, our data strongly support that SST can be used as true marker of AD.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by grants from the Canadian Institutes for Health Research (MOP-6196 and MOP 74465 to UK). Authors are thankful to Dr Brian Cairns for the critical review of the manuscript.


  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • Agren H. and Lundqvist G. (1984) Low levels of somatostatin in human CSF mark depressive episodes. Psychoneuroendocrinology 9, 233248.
  • Banki C. M., Karmacsi L., Bissette G. and Nemeroff C. B. (1992) CSF corticotropin releasing hormone, somatostatin, and thyrotropin releasing hormone in schizophrenia. Psychiatry Res. 43, 1321.
  • Barnea A., Roberts J. and Ho R. H. (1999) Evidence for a synergistic effect of the HIV-1 envelope protein gp120 and brain-derived neurotrophic factor (BDNF) leading to enhanced expression of somatostatin neurons in aggregate cultures derived from the human fetal cortex. Brain Res. 815, 349357.
  • Beal M. F., Mazurek M., Tran V., Chattha G., Bird E. and Martin J. (1985) Reduced number of somatostatin receptors in cerebral cortex in Alzheimer’s disease. Science 229, 289291.
  • Beal M. F., Uhl G., Masurek M. F., Kowall N. and Martin J. B. (1986) Somatostatin: alterations in the central nervous system in neurological diseases, in Neuropeptide in Neurological and Psychiatric Disease (MartinJ. B. and MarchasJ. D., eds), pp. 215254. Raven Press, New York.
  • Beal M. F., Clevens R. A. and Mazurek M. F. (1988) Somatostatin and neuropeptide Y immunoreactivity in Parkinson’s disease dementia with Alzheimer’s changes. Synapse 2, 463467.
  • Bisette G., Cook L., Smith W., Dole K. C., Crain B. and Nemeroff C. B. (1998) Regional neuropeptide pathology in Alzheimer’s disease: corticotropin-releasing factor and somatostatin. J. Alzheimers Dis. 1, 91105.
  • Davies P., Katzman R. and Terry R. D. (1980) Reduced somatostatin-like immunoreactivity in cerebral cortex from cases of Alzheimer disease and Alzheimer senile dementia. Nature 288, 279280.
  • Davis K. L., Mohs R. C., Marin D. B., Purohit D. P., Perl D. P., Lantz M., Austin G. and Haroutunian V. (1999) Neuropeptide abnormalities in patients with early Alzheimer disease. Arch. Gen. Psychiatry 56, 981987.
  • Dawbarn D., DeQuidt M. E. and Emson P. C. (1985) Survival of basal ganglia neuropeptide Y–somatostatin neurons in Huntington’s disease. Brain Res. 340, 251260.
  • Dournaud P., Cervera-Pierot P., Hirsch E., Javoy-Agid F., Kordon C., Agid Y. and Epelbaum J. (1994) Somatostatin messenger RNA-containing neurons in Alzheimer’s disease: an in situ hybridization study in hippocampus, parahippocampal cortex and frontal cortex. Neuroscience 61, 755764.
  • Ferrante R. J., Kowall N. W., Beal M. F., Martin J. B., Bird E. D. and Richardson E. P. (1987) Morphologic and histochemical characteristics of a spared subset of striatal neurons in Huntington’s disease. J. Neuropathol. Exp. Neurol. 46, 1227.
  • Forloni G., Lucca E., Angeretti N., Chiesa R. and Vezzani A. (1997) Neuroprotective effect of somatostatin on non-apoptotic NMDA-induced neuronal death: role of cyclic GMP. J. Neurochem. 68, 319327.
  • Geci C., Glinka Y., Kumar U. and Patel Y. C. (2000) Induction of somatostatin (SST) by β-amyloid peptide in cultured cortical neurons. Program Annual Meeting Society for Neuroscience, New Orleans, LA, November 4–9, 2000.
  • Gonzalez G. A., Yamamoto K. K., Fischer W. H., Karr D., Menzel P., Biggs W. III, Vale W. W. and Montminy M. R. (1989) A cluster of phosphorylation sites on the cyclic AMP-regulated nuclear factor CREB predicted by its sequence. Nature 337, 749752.
  • Grouselle D., Winsky-Sommerer R., David J. P., Delacourte A., Dournaud P. and Epelbaum J. (1998) Loss of somatostatin like immunoreactivity in the frontal cortex of Alzheimer patients carrying the apolipoprotein epsilon 4 allele. Neurosci. Lett. 255, 2124.
  • Ham J., Rickards C. and Scanlon M. (1991) Prolonged exposure to N-methyl-d-aspartate increases intracellular and secreted somatostatin in rat cortical cells. Neurosci. Lett. 129, 262264.
  • Ham J., Duberley R., Rickards C. and Scanlon S. (1993) Differential responses of rat cerebral somatostatinergic and cholinergic cells to glutamate agonists. Mol. Chem. Neuropathol. 19, 107120.
  • Hayashi M., Yamashita A. and Shimizu K. (1997) Somatostatin and brain derived neurotrophic factor mRNA expression in the primate brain: decreased levels of mRNA during aging. Brain Res. 749, 283289.
  • Koutkia P., Meininger G., Canavan B., Breu J. and Grinspoon S. (2004) Metabolic regulation of growth hormone by free fatty acids, somatostatin, and ghrelin in HIV-lipodystrophy. Am. J. Physiol. Endocrinol. Metab. 286, E296E303.
  • Krantic S., Robitaille Y. and Quirion R. (1992) Deficits in the somatostatin SS1 receptor subtype in frontal and temporal cortices in Alzheimer’s disease. Brain Res. 573, 299304.
  • Kumar U. (2004) Characterization of striatal cultured neurons in QUIN and NMDA induced toxicity. Neurosci. Res. 49, 2938.
  • Kumar U. (2005) Expression of somatostatin receptor subtypes (SSTR1–5) in Alzheimer’s disease (AD) brain: an immunohistochemical analysis. Neuroscience 134, 525538.
  • Kumar U., Asotra K., Patel S. C. and Patel Y. C. (1997) Expression of NMDA receptor 1 (NR1) and Huntington in striatal neurons which co-localize somatostatin, neuropeptide Y, and NADPH diaphorase: a double-label histochemical and immunohistochemical study. Exp. Neurol. 145, 412424.
  • Lorenzo A. and Yankner B. A. (1994) Beta-amyloid neurotoxicity requires fibril formation and is inhibited by Congo red. Proc. Natl Acad. Sci. USA 91, 12 24312 247.
  • Lu T., Pan Y., YuanKao S., Li C., Kohane I., Chan J. and Yanker B. A. (2004) Gene regulation and DNA damage in the ageing human brain. Nature 429, 883891.
  • Lynch C. and Mobley W. (2000) Comprehensive theory of Alzheimer’s disease. The effects of cholesterol on membrane receptor trafficking. Ann. N. Y. Acad. Sci. 924, 104111.
  • Nemeroff C. B. (1999) The preeminent role of neuropeptide systems in the early pathophysiology of Alzheimer’s disease: up with corticotropin-releasing factor, down with acetylcholine. Arch. Gen. Psychiatry 56, 991992.
  • Patel S. C., Papachristou D. N. and Patel Y. C. (1991) Quinolinic acid stimulates somatostatin gene expression in cultured rat cortical neurons. J. Neurochem. 56, 12861291.
  • Patel Y. C., Liu J. L., Warszynska A., Kent G., Papachristou D. N. and Patel S. C. (1995) Differential stimulation of somatostatin but not neuropeptide Y gene expression by quinolinic acid in cultured cortical neurons. J. Neurochem. 65, 9981006.
  • Rage F., Rougeot C. and Tapia-Arancibia L. (1994) GABAA and NMDA receptor activation controls somatostatin messenger RNA expression in primary cultures of hypothalamic neurons. Neuroendocrinology 60, 470476.
  • Reichlin S. (1983) Somatostatin. N. Engl. J. Med. 309, 14951501.
  • Sato T., Iwata N., Tsubuki S., Takaki Y., Takano J., Huang S. M., Suemoto T., Higuchi M. and Saido T. C. (2004) Somatostatin regulates brain amyloid B peptide AB42 through modulation of proteolytic degradation. Nat. Med. 11, 434439.
  • Tapia-Arancibia L. and Astier H. (1989) Actions of excitatory amino-acids on somatostatin release from cortical neurons in primary cultures. J. Neurochem. 53, 11341141.
  • Vecsei L., Csala B., Widerlov E., Ekman R., Czopf J. and Palffy G. (1990) Lumbar cerebrospinal fluid concentrations of somatostatin and neuropeptide Y in multiple sclerosis. Brain Res. Bull. 25, 411413.
  • Williams J. S., Berbekar I. and Weiss S. (1991) N-Methyl-d-aspartate evokes the release of somatostatin from striatal interneurons in primary culture. Neuroscience 43, 437444.
  • Wood P. L., Etienne P., Lal S., Gauthier S., Cajal S. and Nair N. P. (1982) Reduced lumbar CSF somatostatin levels in Alzheimer’s disease. Life Sci. 31, 20732079.
  • Yankner B. A., Duffy L. K. and Kirshner D. A. (1990) Neurotrophic and neurotoxic effects of amyloid β protein: reversal by tachykinin neuropeptides. Science 250, 279282.