Daniel P. McCloskey

Chronic administration of corticosterone impairs spatial reference memory before spatial working memory in rats

Corticosterone (CORT), the predominant glucocorticoid in rodents, elevated for 21 days damages hippocampal subregion CA3. We tested the hypothesis that CORT would impair spatial memory, a hippocampal function. In each of the three experiments, rats received daily, subcutaneous injections of either CORT (26.8 mg/kg body weight in sesame oil) or sesame oil vehicle alone (VEH). CORT given for 21 or 56 days effectively attenuated body weight gain and reduced selective organ and muscle weights. All behavioral testing was done on tasks that are minimally stressful and avoid deprivation. For each experiment, testing commenced 24h after the last injection. CORT given for 21 days did not impair spatial working memory in the Y-maze (Experiments 1 and 2). After 56-day administration of CORT, spatial working memory was impaired in the Y-maze (Experiment 2). CORT given for 21 days also failed to impair spatial working memory in the Barnes maze (Experiment 3). However, in trials that depended solely on reference memory, the VEH group improved in performance, whereas the CORT group did not. In conclusion, CORT elevated over a period of 21 days did not impair spatial working memory, but impaired the formation of a longer-term form of memory, most likely reference memory. Impairments in spatial working memory are seen only after longer durations of CORT administration.

Depression of Synaptic Transmission by Vascular Endothelial Growth Factor in Adult Rat Hippocampus and Evidence for Increased Efficacy after Chronic Seizures

In addition to its potent effects on vasculature, it has become clear that vascular endothelial growth factor (VEGF) has effects on both neurons and glia, and recent studies suggest that it can be neuroprotective. To determine potential mechanisms underlying this neuroprotection, recombinant human VEGF was bath applied to adult rat hippocampal slices, and both extracellular and intracellular recordings were used to examine intrinsic properties and synaptic responses of hippocampal principal neurons. Initial studies in area CA1 showed that VEGF significantly reduced the amplitude of responses elicited by Schaffer collateral stimulation, without influencing membrane properties. Similar effects occurred in CA3 pyramidal cells and dentate gyrus granule cells when their major glutamatergic afferents were stimulated. Because VEGF expression is increased after seizures, effects of VEGF were also examined in rats with recurrent spontaneous seizures. VEGF reduced spontaneous discharges in slices from these rats but had surprisingly little effect on epileptiform discharges produced by disinhibition of slices from control rats. These results demonstrate a previously unknown effect of VEGF on neuronal activity and also demonstrate a remarkable potency in the epileptic brain. Based on this, we suggest that VEGF or VEGF-related targets could provide useful endpoints to direct novel therapeutic strategies for epilepsy.

Stereological methods reveal the robust size and stability of ectopic hilar granule cells after pilocarpine-induced status epilepticus in the adult rat

Following status epilepticus in the rat, dentate granule cell neurogenesis increases greatly, and many of the new neurons appear to develop ectopically, in the hilar region of the hippocampal formation. It has been suggested that the ectopic hilar granule cells could contribute to the spontaneous seizures that ultimately develop after status epilepticus. However, the population has never been quantified, so it is unclear whether it is substantial enough to have a strong influence on epileptogenesis. To quantify this population, the total number of ectopic hilar granule cells was estimated using unbiased stereology at different times after pilocarpine‐induced status epilepticus. The number of hilar neurons immunoreactive for Prox‐1, a granule‐cell‐specific marker, was estimated using the optical fractionator method. The results indicate that the size of the hilar ectopic granule cell population after status epilepticus is substantial, and stable over time. Interestingly, the size of the population appears to be correlated with the frequency of behavioral seizures, because animals with more ectopic granule cells in the hilus have more frequent behavioral seizures. The hilar ectopic granule cell population does not appear to vary systematically across the septotemporal axis, although it is associated with an increase in volume of the hilus. The results provide new insight into the potential role of ectopic hilar granule cells in the pilocarpine model of temporal lobe epilepsy.

Exercise increases metabolic capacity in the motor cortex and striatum, but not in the hippocampus

Acute bouts of exercise have been shown to produce transient increases in regional cerebral glucose utilization, oxygen uptake, and cerebral blood flow in motor cortex, striatum, and hippocampus. The purpose of this study was to determine whether or not chronic exercise will cause long-term metabolic plasticity in brain structures activated during physical activity. The activity of cytochrome oxidase (COX), is coupled to the production of ATP, and reflects long-term plasticity in metabolic capacity. The present study examined whether or not 6 months of voluntary exercise would increase COX activity in the striatum, sensorimotor cortex, and three hippocampal subfields. Five-month-old, female Long-Evans hooded rats were randomly assigned to a control or exercise condition. Exercising rats had running wheels attached to their home cages. After the training period, fresh brains were rapidly frozen and sectioned with a cryostat. COX activity was measured using COX histochemical methods and optical densitometry. Rats in the exercise condition had significantly higher optical density in the hindlimb and forelimb motor cortices (18%, P<0.01) and dorsolateral caudate putamen (17%, P<0.01), but not in the ventrolateral caudate putamen or any subfield of the hippocampus. Although exercise is believed to increase neuronal activity in the hippocampus, motor cortex and striatum, only limb representations in the motor cortex and striatum increase bioenergetic capacity after regular exercise.

Mossy fibers are the primary source of afferent input to ectopic granule cells that are born after pilocarpine-induced seizures.

Granule cell (GC) neurogenesis increases following seizures, and some newborn GCs develop in abnormal locations within the hilus. These ectopic GCs (EGCs) display robust spontaneous and evoked excitatory activity. However, the pattern of afferent input they receive has not been fully defined. This study used electron microscopic immunolabeling to quantitatively evaluate mossy fiber (MF) input to EGCs since MFs densely innervate the hilus normally and undergo sprouting in many animal models of epilepsy. EGC dendrites were examined in tissue from epileptic rats that had initially been treated with pilocarpine to induce status epilepticus and subsequently had spontaneous seizures. MF terminals were labeled with a zinc transporter-3 antibody, and calbindin immunoreactivity was used to label hilar EGCs and GC layer GCs. The pattern of input provided by sprouted MF terminals to EGC dendrites was then compared to the pattern of MF input to GC dendrites in the inner molecular layer (IML), where most sprouted fibers are thought to project. Analysis of EGC dendrites demonstrated that MF terminals represented their predominant source of afferent input: they comprised 63% of all terminals and, on average, occupied 40% and 29% of the dendritic surface in the dorsal and ventral dentate gyrus, respectively, forming frequent synapses. These measures of connectivity were significantly greater than comparable values for MF innervation of GC dendrites located in the IML of the same tissue sections. Thus, EGCs develop a pattern of synaptic connections that could help explain their previously identified predisposition to discharge in epileptiform bursts and suggest that they play an important role in the generation of seizure activity in the dentate gyrus.

Brain IL-6 elevation causes neuronal circuitry imbalances and mediates autism-like behaviors

Abnormal immune responses have been reported to be associated with autism. A number of studies showed that cytokines were increased in the blood, brain, and cerebrospinal fluid of autistic subjects. Elevated IL-6 in autistic brain has been a consistent finding. However, the mechanisms by which IL-6 may be involved in the pathogenesis of autism are not well understood. Here we show that mice with elevated IL-6 in the brain display many autistic features, including impaired cognitive abilities, deficits in learning, abnormal anxiety traits and habituations, as well as decreased social interactions. IL-6 elevation caused alterations in excitatory and inhibitory synaptic formations and disrupted the balance of excitatory/inhibitory synaptic transmissions. IL-6 elevation also resulted in an abnormal change in the shape, length and distributing pattern of dendritic spines. These findings suggest that IL-6 elevation in the brain could mediate autistic-like behaviors, possibly through the imbalances of neural circuitry and impairments of synaptic plasticity.

Changes in hippocampal function of ovariectomized rats after sequential low doses of estradiol to simulate the preovulatory estrogen surge

In adult female rats, robust hippocampal changes occur when estradiol rises on the morning of proestrus. Whether estradiol mediates these changes, however, remains unknown. To address this issue, we used sequential injections of estradiol to simulate two key components of the preovulatory surge: the rapid rise in estradiol on proestrous morning, and the slower rise during the preceding day, diestrus 2. Animals were examined mid‐morning of simulated proestrus, and compared to vehicle‐treated or intact rats. In both simulated and intact rats, CA1‐evoked responses were potentiated in hippocampal slices, and presynaptic mechanisms appeared to contribute. In CA3, multiple population spikes were evoked in response to mossy fiber stimuli, and expression of brain‐derived neurotrophic factor was increased. Simulation of proestrous morning also improved performance on object and place recognition tests, in comparison to vehicle treatment. Surprisingly, effects on CA1‐evoked responses showed a dependence on estradiol during simulated diestrus 2, as well as a dependence on proestrous morning. Increasing estradiol above the physiological range on proestrous morning paradoxically decreased evoked responses in CA1. However, CA3 pyramidal cell activity increased further, and became synchronized. Together, the results confirm that physiological estradiol levels are sufficient to profoundly affect hippocampal function. In addition: (i) changes on proestrous morning appear to depend on slow increases in estradiol during the preceding day; (ii) effects are extremely sensitive to the peak serum level on proestrous morning; and (iii) there are striking subfield differences within the hippocampus.

Chronic corticosterone affects brain weight, and mitochondrial, but not glial volume fraction in hippocampal area CA3.

Corticosterone (CORT), the predominant glucocorticoid in rodents, is known to damage hippocampal area CA3. Here we investigate how that damage is represented at the cellular and ultrastructural level of analyses. Rats were injected with CORT (26.8 mg/kg, s.c.) or vehicle for 56 days. Cell counts were estimated with the physical disector method. Glial and mitochondrial volume fractions were obtained from electron micrographs. The effectiveness of the CORT dose used was demonstrated in two ways. First, CORT significantly inhibited body weight gain relative to vehicles. Second, CORT significantly reduced adrenal gland, heart and gastrocnemius muscle weight. Both the adrenal and gastrocnemius muscle weight to body weight ratios were also significantly reduced. Although absolute brain weight was reduced, the brain to body weight ratio was higher in the CORT group relative to vehicles, suggesting that the brain is more resistant to the effects of CORT than many peripheral organs and muscles. Consistent with that interpretation, CORT did not alter CA3 cell density, cell layer volume, or apical dendritic neuropil volume. Likewise, CORT did not significantly alter glial volume fraction, but did reduce mitochondrial volume fraction. These findings highlight the need for ultrastructural analyses in addition to cellular level analyses before conclusions can be drawn about the damaging effects of prolonged CORT elevations. The relative reduction in mitochondria may indicate a reduction in bioenergetic capacity that, in turn, could render CA3 vulnerable to metabolic challenges.

A Rat Model of Epilepsy in Women: A Tool to Study Physiological Interactions Between Endocrine Systems and Seizures

Epilepsy in women is influenced by endocrine status and antiepileptic drugs, but without an animal model, the effects of endocrine variables and antiepileptic drugs cannot be easily dissociated from the influence of epilepsy itself. Animal models have had limited utility because experimentally induced seizures typically result in reproductive failure. This study was conducted to develop an improved animal model. The muscarinic convulsant pilocarpine was used to elicit status epilepticus (SE) in adult female Sprague Dawley rats. The selective estrogen receptor modulator raloxifene was administered 30 min before pilocarpine. An anticonvulsant barbiturate, pentobarbital, was injected 5-10 min after the onset of SE and at least once thereafter to minimize acute convulsions. Mortality, morbidity, estrous cyclicity, and the ultimate success of the procedure (i.e. induction of recurrent, spontaneous seizures) were monitored. The combination of raloxifene and pentobarbital led to significantly improved estrous cyclicity compared with previous methods. Animals treated with raloxifene and pentobarbital became epileptic, as defined by the recurrence of spontaneous convulsions in the weeks after SE. The results of this study provide an improved animal model to examine the interactions between seizures and ovarian hormone secretion. The results also suggest that treatment of SE with raloxifene may benefit women with SE.

Modulation of vascular endothelial growth factor (VEGF) expression in motor neurons and its electrophysiological effects

Previous studies have shown that VEGF expression in forebrain increases after experimental manipulations that increase neuronal activity. One question is whether this also occurs in motor neurons. If so, it could be potentially advantageous from a therapeutic perspective, because VEGF prevents motor neuron degeneration. Therefore, we asked whether endogenous VEGF expression in motor neurons could be modulated. We also asked how VEGF exposure would influence motor neurons using electrophysiology. Immunocytochemistry showed that motor neuron VEGF expression increased after a stimulus that increases neuronal and motor activity, i.e., convulsive seizures. The increase in VEGF immunoreactivity occurred in all motor neuron populations that were examined 24h later. This effect was unlikely to be due to seizure-induced toxicity, because silver degeneration stain did not show the typical appearance of a dying or dead neuron. To address the effects of VEGF on motor neuron function, VEGF was applied directly to motor neurons while recording intracellularly, using a brainstem slice preparation. Exposure to exogenous VEGF (200 ng/ml) in normal conditions depressed stimulus-evoked depolarization of hypoglossal motor neurons. There was no detectable effect of VEGF on membrane properties or firing behavior. We suggest that VEGF is upregulated in neurons when they are strongly activated, and VEGF depresses neuronal excitation as a compensatory mechanism. Failure of this mechanism may contribute to diseases that involve a dysregulation of VEGF, excessive excitation of motor neurons, and motor neuron loss, such as amyotrophic lateral sclerosis (ALS).