Cerebellar Purkinje Cell Loss Research Articles

Glial-derived neurotrophic factor (GDNF) prevents ethanol-induced apoptosis and JUN kinase phosphorylation

Ethanol exposure during neural development leads to substantial neuronal loss in multiple brain regions. Our previous research indicated that exogenous glial-derived neurotrophic factor (GDNF) attenuated ethanol-induced cerebellar Purkinje cell loss. Additionally, ethanol decreased GDNF release suggesting that ethanol disrupts GDNF-signaling pathways. The present experiments utilized a homogeneous GDNF-responsive neuroblastoma cell line (SK-N-SH) to test the hypothesis that exogenous GDNF could attenuate ethanol-induced cell loss by suppressing cytotoxic signaling pathways and cell suicide. We measured two independently regulated markers of apoptosis, DNA fragmentation and the externalization of phosphatidylserine to the outer cell membrane leaflet. Ethanol induced a dose-related increase in both apoptosis and necrosis. Lower concentrations of ethanol (34 and 68 mM) specifically increased DNA fragmentation, while all concentrations (up to 137 mM) increased phosphatidylserine translocation, suggesting that ethanol induction of apoptosis is not a unitary process. Furthermore, only higher concentrations of ethanol (103 and 137 mM) induced necrosis. Additionally, ethanol specifically induced phosphorylation of c-jun N-terminal-kinase (JNK), a mitogen-activated protein (MAP) kinase selectively associated with apoptosis. In contrast, ethanol did not alter the phosphorylation of another MAP kinase, the extracellular signal-regulated kinases (ERK) that mediate cell survival. Thus, ethanol activated specific intracellular cell death-associated pathways and induced cell death. GDNF, in turn, prevented both ethanol-induced apoptosis and the activation of the death-associated JNK cascade. Therefore, GDNF may regulate multiple pathways to prevent ethanol-induced cell loss.

Cerebellar purkinje cell loss during life span of the heterozygousStaggerer mouse (Rora+/Rorasg) is gender-related

The staggerer mutation causes dysgenesis of the cerebellar cortex in the homozygous mutant (Rora(sg)/Rora(sg)). The mutation acts intrinsically within the Purkinje cells (PCs), leading to cytological abnormalities and a severe deficit in the number of these cells. In contrast, in the heterozygous staggerer (Rora(+)/Rora(sg)), the cytoarchitecture of the cerebellar cortex appears to be normal, but quantitative studies have revealed a significant loss of cerebellar neurons with advancing age. In the heterozygous reeler (+/rl), another mutant presenting a PC loss with age, we have found that only males were affected (Hadj-Sahraoui et al., 1996). In the present study, we have investigated whether a similar gender effect exists in the heterozygous staggerer during life span. PCs were counted on cerebellar sagittal sections in male and female Rora(+)/Rora(sg) and in their Rora(+)/Rora(+) littermates at 1, 3, 9, 13, 18, and 24 months of age. In the Rora(+)/Rora(+), the number of PCs remained stable until 18 months, but there was a 25% significant loss in 24- month-old mice of both genders. During life span, Rora(+)/Rora(+) males had slightly more PC than females. In the Rora(+)/Rora(sg) of both genders, the deficit in PC number was similar at 13 months but it appeared earlier in males, beginning between 1 and 3 months, and was aggravated regularly up to 13 months. By contrast, the decline was delayed and more abrupt in Rora(+)/Rora(sg) females, from a value still normal at 9 months to its maximal extent at 13 months. In view of these results, the heterozygous (Rora(+)/Rora(sg)) mouse offers an interesting model to test the interaction between sex, age, and genetic background on the development and maintenance of cerebellar neuronal populations.

Cerebellar purkinje cell loss during life span of the heterozygous Staggerer mouse (RoraRorasg) is gender‐related

The staggerer mutation causes dysgenesis of the cerebellar cortex in the homozygous mutant (Rorasg/Rorasg). The mutation acts intrinsically within the Purkinje cells (PCs), leading to cytological abnormalities and a severe deficit in the number of these cells. In contrast, in the heterozygous staggerer (Rora+/Rorasg), the cytoarchitecture of the cerebellar cortex appears to be normal, but quantitative studies have revealed a significant loss of cerebellar neurons with advancing age. In the heterozygous reeler (+/rl), another mutant presenting a PC loss with age, we have found that only males were affected (Hadj-Sahraoui et al., 1996). In the present study, we have investigated whether a similar gender effect exists in the heterozygous staggerer during life span. PCs were counted on cerebellar sagittal sections in male and female Rora+/Rorasg and in their Rora+/Rora+ littermates at 1, 3, 9, 13, 18, and 24 months of age. In the Rora+/Rora+, the number of PCs remained stable until 18 months, but there was a 25% significant loss in 24- month-old mice of both genders. During life span, Rora+/Rora+ males had slightly more PC than females. In the Rora+/Rorasg of both genders, the deficit in PC number was similar at 13 months but it appeared earlier in males, beginning between 1 and 3 months, and was aggravated regularly up to 13 months. By contrast, the decline was delayed and more abrupt in Rora+/Rorasg females, from a value still normal at 9 months to its maximal extent at 13 months. In view of these results, the heterozygous (Rora+/Rorasg) mouse offers an interesting model to test the interaction between sex, age, and genetic background on the development and maintenance of cerebellar neuronal populations. J. Comp. Neurol. 411:267–273, 1999. © 1999 Wiley-Liss, Inc.

Alcohol-induced Purkinje cell loss depends on developmental timing of alcohol exposure and correlates with motor performance

Several reports indicate that neonatal ethanol exposure induces cerebellar Purkinje and granule cell loss if exposure occurs before postnatal day (PD) 7, and that cerebellar damage may underlie ethanol-induced motor deficits. The present study used an unbiased stereological method, the optical fractionator, to count total cerebellar Purkinje cell number in groups of Sprague–Dawley rats given binge-like ethanol exposure at different neonatal ages. Correlations between Purkinje cell number (of 55-day-old rats) and parallel bar motor performance (previously tested on PD 30–32) were also evaluated. One group was given binge-like exposure to 6.6 g/kg per day of ethanol via artificial rearing on PD 4 and 5 (PD 4/5); a second group on PD 8 and 9 (PD 8/9); and a third group on both PD 4 and 5 and 8 and 9 (Comb). Gastrostomy (GC) and suckle (SC) control groups were also included. Purkinje cells were significantly reduced in all three ethanol-treated groups compared to controls, but the severity of loss was significantly greater in the PD 4/5 and Comb groups (reduced by 42% and 45%, respectively, relative to GC) compared to the PD 8/9 group (reduced by 15%). Across treatment groups, the total cerebellar Purkinje cell number was significantly correlated with successful parallel bar traversal ( r=+0.74), supporting the contention that ethanol-induced motor deficits may be associated with cerebellar cell loss. These data confirm the presence of windows of vulnerability of Purkinje cells to neurotoxic effects of binge ethanol treatment, and demonstrate that both the behavioral and neuroanatomical consequences of binge exposure depend on the developmental timing of the exposure.

Alcohol‐Induced Purkinje Cell Loss with a Single Binge Exposure in Neonatal Rats: A Stereological Study of Temporal Windows of Vulnerability

Previous research has shown that the early neonatal period of rats is one of enhanced vulnerability to cerebellar Purkinje cell loss associated with binge-like alcohol exposure, with a prominent sensitive period during the first neonatal week. In this study, an unbiased count of the total number of Purkinje cells was obtained using the stereological optical fractionator, in groups of rats given a single binge-like alcohol exposure either during the most vulnerable neonatal period [postnatal day (PD) 4] or during a later, less vulnerable period (PD 9). Using artificial rearing methods, rats were given 6.6 g/kg of alcohol either on PD 4 or on PD 9, delivered as a 15% (v/v) solution in milk formula on two consecutive feedings of the designated day. Control groups included an artificially reared gastrostomy control and a normally reared suckle control. The mean peak blood alcohol concentrations were not different between the PD 4 and PD 9 alcohol groups, averaging 374 and 347 mg/dl, respectively. The rats were perfused on PD 27. A uniform random sample of sections was obtained from serial frozen sections through the cerebellum, stained with thionin, and Purkinje cells were counted from a uniform random sample of locations on each section with the three-dimensional optical fractionator. The number of Purkinje cells in the suckle control and gastrostomy control groups did not differ from each other, averaging 3.94 (+/- 0.19) and 3.58 (+/- 0.22) x 10(5) cells, respectively. Binge exposure on PD 4 induced significant cell loss (mean of 2.05 +/- 0.20 x (10(5) Purkinje cells), whereas binge exposure on PD 9 did not induce significant Purkinje cell loss (3.70 +/- 0.39 x 10(5) Purkinje cells). These findings confirm that a single neonatal binge alcohol exposure produces pathological Purkinje cell loss, provided that it occurs during the period of enhanced vulnerability coinciding with the early stages of dendritic outgrowth.

Cocaine exposure during the brain growth spurt failed to produce cerebellar Purkinje cell loss in rat pups.

Previous studies in our laboratory indicated that cocaine exposure during the brain growth spurt period, a developmental stage vulnerable to various teratogens, did not produce microencephaly (gross brain weight measures). However, neonatal cocaine exposure has been shown to affect motor coordination and balance, which are both sensitive to cerebellar damage. The purpose of this study was to investigate whether cocaine exposure during the brain growth spurt period could result in the loss of cerebellar Purkinje cells, a neuronal population known to be vulnerable to other teratogenic insults. Sprague-Dawley rat pups were randomly assigned to either cocaine-treated groups (40, 80 mg/kg s.c.) or a gastrostomy control group, and were reared using an artificial-rearing method from postnatal days (PDs) 4 through 9. On PD 10, these animals were perfused and the cerebella were extracted and processed for cell counts. Estimates of Purkinje cell numbers were obtained using a 3-dimensional optical dissector method. The results using this stereological method demonstrated no significant Purkinje cell loss in response to cocaine treatment, even at a dose which has been shown to result in high mortality. The failure of cocaine to produce significant Purkinje cell loss (present finding) or microencephaly (previous finding) odds to the evidence indicating that cocaine is not a potent neuroteratogen.

Purkinje cell loss from alternating sagittal zones in the cerebellum of leaner mutant mice

The cerebellum and inferior olive of adult homozygous leaner mutant mice ( tg la/ tg la) were examined using anti-vitamin D-dependent calcium binding protein immunohistochemistry, and Nissl staining. The immunohistochemical staining revealed that cerebellar Purkinje cell loss in leaner is restricted to alternating sagittal compartments of the cerebellar cortex. The absence of Purkinje cells from specific sagittal zones of the cerebellar cortex apparently results in a loss of immunochemical staining of Purkinje cell axons and terminals from specific regions of the leaner cerebellar nuclear complex. A specific pattern of atrophy was also observed in the leaner inferior olive.

The AMPA antagonist, NBQX, protects against ischemia-induced loss of cerebellar Purkinje cells.

We examined the effect of an AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole) antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline (NBQX), on rat cerebellar Purkinje cell loss and hippocampal pyramidal CA1 cell loss, after 10 minutes of global cerebral ischemia. NBQX was given intraperitoneally in a dose of 30 mg kg-1 at the end of ischemia, and 10 and 25 minutes later. Rats subjected to ischemia without post-ischemic administration of NBQX served as controls. Four days after ischemia the cerebellar Purkinje cell density was higher and the density of acidophilic (dead) Purkinje cells lower in the NBQX treated animals compared with the control animals (p = 0.01 and p less than 0.005 respectively). There was partial to total loss of pyramidal neurons in the CA1 region of the dorsal hippocampus in control animals, but no CA1 pyramidal neuron loss in the NBQX treated animals (p = 0.001).

A single day of alcohol exposure during the brain growth spurt induces brain weight restriction and cerebellar Purkinje cell loss

The period of rapid brain growth that occurs relatively late in development has been shown to be vulnerable to alcohol-induced brain growth deficits and neuron loss in rats using repeated daily exposure to alcohol. This study examined whether a condensed exposure (binge) restricted to a single day during this period would be sufficient to restrict brain growth and produce neuron depletion. Using artificial rearing, alcohol was given to neonatal rats in two consecutive feedings on postnatal day 4, using a total dose of either 6.6 g/kg or 3.3 g/kg. The higher dose produced mean peak blood alcohol concentrations of 362 mg/dl, while the lower dose produced mean peak BACs of 153 mg/dl. The higher dose significantly restricted whole brain, forebrain, cerebellum and brain stem weights on postnatal day 10, with the cerebellum being most severely reduced. Cerebellar Purkinje cells, counted from a 2-μm section from the midsagittal vermis, were significantly reduced in the 6.6 g/kg group. Purkinje cell loss relative to controls was significant in most lobules (I–V, IX and X), but, notably, was not significant in the later maturing regions (lobules VI and VII). The 3.3 g/kg group had no significant reductions in regional brain weights. Although the overall number of Purkinje cells was nonsignificantly reduced relative to controls, the 3.3 g/kg dose did produce significant loss of Purkinje cells in lobule II. Exposure to high peak BACs, even for a relatively short period during the brain growth spurt, constitutes a substantial risk to the developing brain, and even a moderate exposure may result in loss of more vulnerable neurons. The pattern of cell death produced suggests that the maturational or metabolic state of Purkinje cells at the time of the alcohol insult may influence the vulnerability of these neurons to an alcohol insult.