Dorothy E. Oorschot

Total number of neurons in the neostriatal, pallidal, subthalamic, and substantia nigral nuclei of the rat basal ganglia: A stereological study using the cavalieri and optical disector methods

The total number of neurons within six subdivisions of the rat basal ganglia was estimated using unbiased stereological counting methods and systematic random sampling techniques. Six young adult rats were perfuse‐fixed, their right cerebral hemispheres were embedded in glycolmethacrylate, and a complete set of serial 40‐μm sections was cut through each hemisphere. After a random start, a systematic subset (e.g., every tenth) of these sections was used to estimate the total volume of each subdivision using Cavalieris method. The same set of sampled sections was used to estimate the number of neurons in a known subvolume (i.e., the Nv) by the optical disector method. The product of the total volume and the Nv by these methods yields an unbiased estimate of the total number of neurons. It was found that the right basal ganglia consisted, on average, of 2.79 million neostriatal or caudate‐putamen neurons (with a coefficient of variation of 0.07), 46,000 external globus pallidus neurons (0.11), 3,200 entopeduncular/internal globus pallidus neurons (0.10), 13,600 subthalamic neurons (0.10), 7,200 substantia nigra pars compacta neurons (0.15), and 26,300 substantia nigra pars reticulata neurons (0.07).

Cell loss in the motor and cingulate cortex correlates with symptomatology in Huntington’s disease

Huntingtons disease is an autosomal dominant inherited neurodegenerative disease with motor symptoms that are variably co-expressed with mood and cognitive symptoms, and in which variable neuronal degeneration is also observed in the basal ganglia and the cerebral cortex. We have recently shown that the variable symptomatology in Huntingtons disease correlates with the variable compartmental pattern of GABAA receptor and cell loss in the striatum. To determine whether the phenotypic variability in Huntingtons disease is also related to variable neuronal degeneration in the cerebral cortex, we undertook a double-blind study using unbiased stereological cell counting methods to determine the pattern of cell loss in the primary motor and anterior cingulate cortices in the brains of 12 cases of Huntingtons disease and 15 controls, and collected detailed data on the clinical symptomatology of the patients with Huntingtons disease from family members and clinical records. The results showed a significant association between: (i) pronounced motor dysfunction and cell loss in the primary motor cortex; and (ii) major mood symptomatology and cell loss in the anterior cingulate cortex. This association held for both total neuronal loss (neuronal N staining) and pyramidal cell loss (SMI32 staining), and also correlated with marked dystrophic changes in the remaining cortical neurons. There was also an association between cortical cell loss and striatal neuropathological grade, but no significant association with CAG repeat length in the Huntingtons disease gene. These findings suggest that the heterogeneity in clinical symptomatology that characterizes Huntingtons disease is associated with variation in the extent of cell loss in the corresponding functional regions of the cerebral cortex whereby motor dysfunction correlates with primary motor cortex cell loss and mood symptomatology is associated with cell loss in the cingulate cortex.

Continuous low-dose treatment with brain-derived neurotrophic factor or neurotrophin-3 protects striatal medium spiny neurons from mild neonatal hypoxia/ischemia: a stereological study

This study aimed to investigate whether continuous, low-dose, intracerebral infusion of either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) could protect against striatal neuronal loss in mild neonatal hypoxic/ischaemic brain injury. Continuous, low-dose, intracerebral treatment is likely to minimise unwanted side effects of a single high dose and lengthen the time window for neuroprotection. A milder, albeit brain damage-inducing, hypoxic/ischaemic injury paradigm was used since this situation is likely to produce the highest survival rates and thus the greatest prevalence. Anaesthetised postnatal day 7 rats were each stereotaxically implanted with a brain infusion kit connected to a micro-osmotic pump. The pump continuously infused either BDNF (4.5 microg/day), NT-3 (12 microg/day), or vehicle solution into the right striatum for 3 days from postnatal day 7. The intrastriatal presence of BDNF or NT-3 was verified immunohistochemically. On postnatal day 8, the rats underwent right common carotid artery ligation followed by hypoxic exposure for 1.5 h. Animals were weighed daily thereafter and killed 1 week later on postnatal day 14. The total number of medium spiny neurons within the right striatum was stereologically determined using an optical disector/Cavalieri combination. Other measures of neuroprotection such as brain weight and striatal infarct volume were also undertaken. BDNF or NT-3 significantly increased the total number of surviving medium spiny neurons by 43% and 33% respectively. This significant neuroprotection was not evident when brain weight, striatal volume, striatal infarct volume, and neuronal density measures for NT-3, were compared. These measures therefore missed the protective effect demonstrated by the total neuronal count. This suggests that stereological measurement of total neuronal number is needed to detect neuroprotection at 1 week after low-dose, continuously infused, neurotrophin treatment and mild hypoxic/ischaemic injury. The results also suggest that lower treatment doses may be more useful than previously thought. BDNF may be particularly useful since it fostered both neuroprotection and normal weight gain. The ability to rescue striatal neurons from death may contribute toward a potential short-term, low-dose neurotrophin treatment for mild perinatal hypoxic/ischaemic brain injury in humans.

Postinjury magnesium sulfate treatment is not markedly neuroprotective for striatal medium spiny neurons after perinatal hypoxia/ischemia in the rat.

Hypoxic/ischemic (H/I) brain injury is thought to be mediated via the N-methyl-D-aspartate receptor complex, which can be blocked by the magnesium ion. Striatal medium spiny neurons abundantly express N-methyl-D-aspartate receptors and are known to be injured after H/I. Thus, the aim of this study was to investigate the effect of postinjury magnesium treatment on the total number of medium spiny neurons in the striatum after perinatal H/I injury in the rat. Anesthetized postnatal day (PN) 7 rats underwent common carotid artery ligation followed 2 h later by exposure to hypoxia for 1.5 h. Contralateral hemispheres served as controls as did animals exposed to normoxia. Immediately after hypoxia or normoxia, the magnesium groups received s.c. injections of 300 mg/kg MgSO4. Control, hypoxic or normoxic animals received NaCl injections. This continued daily until PN13. Eleven matched-for-weight H/I pups were injected in total. A power calculation showed that 11 pups per treatment group would permit detection of a treatment difference of 32% or more. Animals were killed on PN18, and 40-µm serial sections were cut through each entire striatum. The total number of the predominant medium spiny neurons within each striatum was stereologically determined via the use of an unbiased optical dissector/Cavalieri combination. It was found that postinjury magnesium treatment did not improve neuronal survival by 32% or more in the striatum. The results suggest that magnesium treatment after perinatal H/I damage in the rat is not markedly neuroprotective for striatal medium spiny neurons.

The number, size, and type of axons in rat subcortical white matter on left and right sides: a stereological, ultrastructural study.

Abundant evidence indicates important functional differences between the two cerebral hemispheres of humans, although the cellular basis of these differences is unknown. A recent hypothesis proposes that these functional differences depend on differences between sides in the “repertoire” of axonal conduction delays for cortico-cortical axons. In morphological terms this corresponds to differences in caliber, or proportion, of myelinated versus unmyelinated axons. Several behavioural studies have indicated that cerebral asymmetry occurs in rodents, in which rigorous morphological analysis is possible. The hypothesis was therefore tested for the first time in adult male Wistar rats, using transmission electron microscopy and stereological methods. Subcortical white matter was compared between left and right sides in three regions (frontal, parietal, and occipital). The average caliber and numerical density of unmyelinated and myelinated axons was compared between sides and between regions. All data were corrected for shrinkage. No significant differences between sides were found in the average caliber of either type of axon in any region. The numerical density of either type of axon also yielded no significant differences between sides in any region. Significant differences were evident between regions in both caliber and numerical density of the two axonal types, and these quantitative data are reported. The proportion of unmyelinated axons in the lateral white matter was also higher than in previous studies of hemispheric white matter that studied the corpus callosum. The present study provides no evidence supporting the hypothesis that functional hemispheric specialization is due to differences in axonal number, caliber or type.

The trophic requirements of mature motoneurons

Mature motoneurons interact with many cell types, including skeletal muscle fibres, Schwann cells, glia and various neurons. Each of these cell types is thought to provide trophic support to motoneurons, but it is not known whether the support provided by one cell type can fully substitute for the absence of a signal from another cell type. The ability of various growth factors to support motoneurons in the absence of muscle fibres, Schwann cells or long-axon synaptic input has been studied using in vivo models. However, these studies do not define the total needs of motoneurons, as local spinal influences have not been removed. In this paper, the total trophic requirement of mature motoneurons was assessed by culturing them at a low cell density, in the absence of all other cell types. Under these conditions, mature motoneurons die by apoptosis within 24 h, which is equivalent to the rate at which immature motoneurons die in vitro. This is consistent with the emerging view that mature cells are primed for apoptosis. Nine putative trophic factors (BDNF, CNTF, FGF2, GDNF, IGF I, IGF II, NT3, NT4, TGF-beta2), either alone or in combination, were unable to prevent the rapid death of the cultured motoneurons, even though some of these factors are able to attenuate the affects of less severe injuries such as axotomy or avulsion. The survival of mature motoneurons may therefore be dependent on a combination of growth factors, with at least one of the factors being distinct from the above mentioned factors.

Effect of the mitochondrial antioxidant, Mito Vitamin E, on hypoxic-ischemic striatal injury in neonatal rats: a dose-response and stereological study.

A mitochondria-targeted antioxidant, Mito Vitamin E (MitoVit E), has previously been shown to prevent mitochondrial oxidative damage. The aim of this study was to investigate the effect of MitoVit E on neuronal survival in the rat striatum after acute perinatal hypoxia-ischemia. Continuous striatal infusion with 4.35 microM, 43.5 microM, or 148 microM of MitoVit E before, during, and after hypoxia-ischemia was not neuroprotective for striatal medium-spiny neurons. Pre- or posttreatment with 435 microM MitoVit E was neurotoxic. These results suggest that MitoVit E is not significantly neuroprotective for striatal medium-spiny neurons after acute perinatal hypoxic-ischemic brain injury. The results also suggest that mitochondrial oxidative damage does not contribute significantly to the death of striatal medium-spiny neurons after perinatal hypoxia-ischemia.

IH current generates the afterhyperpolarisation following activation of subthreshold cortical synaptic inputs to striatal cholinergic interneurons

Pauses in the tonic firing of striatal cholinergic interneurons emerge during reward‐related learning and are triggered by neutral cues which develop behavioural significance. In a previous in vivo study we have proposed that these pauses in firing may be due to intrinsically generated afterhyperpolarisations (AHPs) evoked by excitatory synaptic inputs, including those below the threshold for action potential firing. The aim of this study was to investigate the mechanism of the AHPs using a brain slice preparation which preserved both cerebral hemispheres. Augmenting cortically evoked postsynaptic potentials (PSPs) by repetitive stimulation of cortical afferents evoked AHPs that were unaffected by blocking either GABAA receptors with bicuculline, or GABAB receptors with saclofen or CGP55845. Apamin (a blocker of small conductance Ca2+‐activated K+ channels) had minimal effects, while chelation of intracellular Ca2+ with BAPTA reduced the AHP by about 30%. In contrast, blocking hyperpolarisation and cyclic nucleotide activated (HCN) cation current (IH) with ZD7288 or Cs+ diminished the size of the AHPs by 60% and reduced the proportion of episodes that contained this hyperpolarisation. The reversal potential (−20 mV) and voltage dependence of the AHPs were consistent with the hypothesis that a transient deactivation of IH caused most of the AHP at hyperpolarised potentials, while the slow AHP‐type Ca2+‐activated K+ channels increasingly contributed at more depolarised membrane potentials. Subthreshold somatic current injections yielded similar AHPs with a median duration of ∼700 ms that were not affected by firing of a single action potential. These results indicate that transient deactivation of HCN channels evokes pauses in tonic firing of cholinergic interneurons, an event likely to be elicited by augmentation of afferent synaptic inputs during learning.

Cortical Interneuron Loss and Symptom Heterogeneity in Huntington Disease

The cellular basis of variable symptoms in Huntington disease (HD) is unclear. One important possibility is that degeneration of the interneurons in the cerebral cortex, which play a critical role in modulating cortical output to the basal ganglia, might play a significant role in the development of variable symptomatology in HD. This study aimed to examine whether symptom variability in HD is specifically associated with variable degeneration of cortical interneurons.

Widespread Heterogeneous Neuronal Loss Across the Cerebral Cortex in Huntington's Disease

Huntingtons disease is an autosomal dominant neurodegenerative disease characterized by neuronal degeneration in the basal ganglia and cerebral cortex, and a variable symptom profile. Although progressive striatal degeneration is known to occur and is related to symptom profile, little is known about the cellular basis of symptom heterogeneity across the entire cerebral cortex. To investigate this, we have undertaken a double blind study using unbiased stereological cell counting techniques to determine the pattern of cell loss in six representative cortical regions from the frontal, parietal, temporal, and occipital lobes in the brains of 14 Huntingtons disease cases and 15 controls. The results clearly demonstrate a widespread loss of total neurons and pyramidal cells across all cortical regions studied, except for the primary visual cortex. Importantly, the results show that cell loss is remarkably variable both within and between Huntingtons disease cases. The results also show that neuronal loss in the primary sensory and secondary visual cortices relate to Huntingtons disease motor symptom profiles, and neuronal loss across the associational cortices in the frontal, parietal and temporal lobes is related to both Huntingtons disease motor and to mood symptom profiles. This finding considerably extends a previous study (Thu et al., Brain, 2010; 133:1094-1110) which showed that neuronal loss in the primary motor cortex was related specifically to the motor symptom profiles while neuronal loss in the anterior cingulate cortex was related specifically to mood symptom profiles. The extent of cortical cell loss in the current study was generally related to the striatal neuropathological grade, but not to CAG repeat length on the HTT gene. Overall our findings show that Huntingtons disease is characterized by a heterogeneous pattern of neuronal cell loss across the entire cerebrum which varies with symptom profile.