Prenatal ketamine exposure causes abnormal development of prefrontal cortex in rat

Abstract

Ketamine is commonly used for anesthesia and as a recreational drug. In pregnant users, a potential neurotoxicity in offspring has been noted. Our previous work demonstrated that ketamine exposure of pregnant rats induces affective disorders and cognitive impairments in offspring. As the prefrontal cortex (PFC) is critically involved in emotional and cognitive processes, here we studied whether maternal ketamine exposure influences the development of the PFC in offspring. Pregnant rats on gestational day 14 were treated with ketamine at a sedative dose for 2 hrs, and pups were studied at postnatal day 0 (P0) or P30. We found that maternal ketamine exposure resulted in cell apoptosis and neuronal loss in fetal brain. Upon ketamine exposure in utero, PFC neurons at P30 showed more dendritic branching, while cultured neurons from P0 PFC extended shorter neurites than controls. In addition, maternal ketamine exposure postponed the switch of NR2B/2A expression, and perturbed pre- and postsynaptic protein expression in the PFC. These data suggest that prenatal ketamine exposure impairs neuronal development of the PFC, which may be associated with abnormal behavior in offsprings.

Figure 1. Ketamine induces a widespread apoptosis in fetal brain.

Embryonic day 14 coronal sections were stained with TUNEL. In control (a–c) and ketamine (d–f) groups, positive cells were widely distributed in the ventral telencephalon (vTel), hippocampal primordium (Hip) and neocortex (NCx). Positive cells in different brain areas were significantly increased in the ketamine compared to the control group (g): vTel; (h): Hip; (i): NCx). LV, lateral ventricle. *p < 0.05; **p < 0.01; n = 6 in each group.

Figure 2. Caspase-3 positive cells in the fetal brain.

Embryonic day 14 sections were stained with antibodies against cleaved caspase-3. In control (a–c) and ketamine (d–f) groups, positive cells were widely distributed in the ventral telencephalon (vTel), hippocampal primordium (Hip) and neocortex (NCx). Their densities were significantly increased in the ketamine compared to the control group in different areas ((g) vTel; (h) Hip; (i) NCx). LV, lateral ventricle. *p < 0.05; **p < 0.01; n = 6 in each group.

Figure 3. Ketamine causes neuronal loss in laminae II-III of the PFC in offspring.

Coronal sections encompassing the PFC at P0 and P30, stained with cresyl violet. The boxed area indicated in panel a was chosen for analysis. At P0, the cytoarchitecture of the PFC was comparable in control (b) and ketamine (c) groups, but the cell density in laminae II-III was significantly decreased in the ketamine group (h). At P30, cell distribution was similar in laminae II-III (d,e) and V (f,g) of control (d,f) and ketamine-treated animals (e,g). In the ketamine group, the cell density was significantly decreased in laminae II-III (h) but not in lamina V (i), compared to the control group. PrL, prelimbic cortex; *p < 0.05; n = 6 in each group.

Figure 4. Ketamine exposure disturbs the maturation of pyramidal neurons in the PFC of offspring.

Six P30 brains in each group were processed for Golgi–Cox impregnation and pyramidal neurons in laminae II-III of the PFC were studied in the control (examples in a) and ketamine (examples in b) groups. The left panels of (a,b) show two examples of Golgi-impregnated neurons. The total branch number and dendritic length were significantly increased in the ketamine compared to the control group (c,d) n = 60 neurons in each group). In addition, the ketamine group had higher spine density than the control (a’, b’, (e) n = 60 dendrites in each group). Sholl analysis showed that the complexity of dendritic trees was higher in the ketamine than in the control group (f) Kolmogorov–Smirnov test). *p < 0.05; **p < 0.01.

Figure 5. Ketamine administration in utero inhibits neurite growth in neuronal culture.

P0 PFC neurons from pups whose mothers received ketamine at gestational day 14 or not (control) (n = 6 in each group) were cultured for 4 DIV, immunostained with anti-β-tubulin antibodies (a,b), prior to reconstruction using Imaris (examples in (c–g), control; (h–l), ketamine group). Quantitative analysis showed that the total neurite length and number were significantly lower in the ketamine than the control groups ((m,n); n = 60 neurons in each group). This difference was confirmed by Sholl analysis (o). *p < 0.05; **p < 0.01.

Figure 6. Expression of NR2B/2A, PSD-95 and synaptophysin is affected by maternal exposure to ketamine.

PFC tissue from P0 and P30 rats were processed for western blots to detect proteins NR2B (a), NR2A (b), synaptophysin (SY-38, e) and PSD-95 (f). The NR2B protein levels in the ketamine group were significantly lower at P0 and higher at P30 compared to the corresponding control samples (a,c). The NR2A protein was expressed at a low level at P0 in both control and ketamine samples (b,d) but was significantly decreased in the ketamine compared to the control at P30 (b,d). The ketamine group expressed significantly less SY-38 at P0 and P30 relatively to controls (e,g). In contrast, there was no significant difference of PSD-95 levels at P0 and more PSD-95 protein in the ketamine group than in the control at P30 (f,h). ctrl-P0, control group at P0; ctrl-P30, control group at P30; ktm-P0, the ketamine group at P0; ktm-P30, ketamine group at P30. *p < 0.05; **p < 0.01, comparison between the control and ketamine group; ^#p < 0.05; ^##p < 0.01, comparison between P0 and P30; n = 6 in each group.

Figure 7. The numbers of PSD-95 and synaptophysin positive cells in PFC are modified by ketamine exposure.

The rat with location of the medial prefrontal cortex (PrL, a). Areas of prefrontal cortex as indicated in (b) and laminae II-III as indicated in (e–h) were selected for analysis. Paraffin coronal sections from P30 PFC were stained with anti-PSD-95 and anti-synatophysin (SY-38) antibodies. Fluorescent immunohistochemistry for PSD-95 (green) and SY-38 (green), and DAPI (blue). The signal was estimated by quantifying the density of immnofluorescence using Image J. The ketamine group showed a significant increase of PSD-95 immunoreactivity (c) and a significant decrease of synaptophysin (d) at P30 compared to the control. *p < 0.05; n = 6 in each group.

Figure 8. The flow chart of the experimental protocols.