Long-term Effects of Cannabidiol and/or Fentanyl Exposure in Rats Submitted to Neonatal Pain.

The current study aimed to evaluate anxiety behavior, hippocampal ionized calcium-binding adaptor molecule 1 (Iba1) and cannabinoid receptor 1 (CB1) gene expression, and nociceptive response in adulthood after a combination of fentanyl and cannabidiol (CBD) for nociceptive stimuli induced during the first week of life in rats. Complete Freund's adjuvant-induced inflammatory nociceptive insult on postnatal day (PN) 1 and PN3. Both fentanyl and CBD were used alone or in combination from PN1 to PN7. Behavioral and nociceptive tests were performed at PN60 and PN62. The expression of the microglial calcium-binding proteins Iba1 and CB1 was detected in the hippocampus using reverse Quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Our results suggest that the anxiety behavior response and immune activation in adult life depend on the CBD dose combined with fentanyl for the nociceptive stimuli induced during the first week of life. Treatment of neonatal nociceptive insult with CBD and opioids showed significant dose-dependent and male-female differences. The increased gene expression in the hippocampus of the analyzed cannabinoid gene supports this data. In addition, treatment with fentanyl led to an increase in CB1 protein expression. Moreover, the expression of Iba1 varied according to the administered dose of CBD and may or may not be associated with the opioid. A lower dose of CBD during the inflammatory period was associated with enhanced anxiety in adult life. PERSPECTIVE: The treatment of nociceptive stimuli with CBD and opioids during the first week of life demonstrated significant sex differences in adult life on anxiety behavior and supraspinal pain sensitivity.

Anxious Profile Influences Behavioral and Immunohistological Findings in the Pilocarpine Model of Epilepsy.

Anxiety and epilepsy have a complex bidirectional relationship, where a depressive/anxious condition is a factor that can trigger seizures which in turn can aggravate the depressive/anxious condition. In addition, brain structures such as the hippocampus and amygdala might have a critical relevance in both epilepsy and anxiety. The aim of the present work was to investigate the influence of different anxious profiles to epileptogenesis. Initially, animals were screened through the elevated plus-maze anxiety test, and then seizure development was evaluated using the pilocarpine model of epilepsy. There were no differences in the susceptibility to status epilepticus, mortality rate or frequency of spontaneous recurrent seizures between animals characterized as anxious as compared to the non-anxious animals. Next, we evaluated immunohistological patterns related to seizures and anxiety in various related brain areas. Despite a decrease in the density of neuropeptide Y and parvalbumin expression in epileptic animals, those presenting greater neuropeptide Y immunoreactivity in various brain regions, also showed higher spontaneous recurrent seizures frequency. Differences on the anxious profile showed to interfere with some of these findings in some regions. In addition, animals that were injected with pilocarpine, but did not develop status epilepticus, had behavioral and neuroanatomical alterations as compared to control animals, indicating its importance as an additional tool for investigating the heterogeneity of the epileptogenic response after an initial insult. This study allowed to better understand the association between anxiety and temporal lobe epilepsy and might allow for therapeutic targets to be developed to minimize the negative impacts associated with it.

Two decades of research towards a potential first anti-epileptic drug.

The basic mechanisms by which brain insults, such as trauma, stroke or status epilepticus produce epilepsy are not completely understood, and effective preventive measures and treatment are still not available in the clinical setting. Over the last 2 decades we have conducted several studies with animal models of epilepsy (rodents and non-human primates) and demonstrated that drugs that modify neuronal plastic processes, such as anticholinergic agents (e.g., antimuscarinic compounds), if administered soon after brain injury and over a period of 10-20 days, have the potential to modify the natural course of post-traumatic epilepsy. To that end treatment with scopolamine showed promising results as a candidate agent in both the pilocarpine and kainate models. We then showed that biperiden, yet another cholinergic antagonist acting in the muscarinic receptor, that is widely used to treat Parkinson's disease, also decreased the incidence and intensity of spontaneous epileptic seizures, delaying their appearance in the pilocarpine model of epilepsy. In other words, biperiden showed to be a potential candidate to be further investigated as an antiepileptogenic agent. Accordingly, we tested the safety of biperiden in a small group of patients (as a small phase II safety assessment) and confirmed its safety in the context of traumatic brain injury (TBI). Now, we provide information on our ongoing project to evaluate the efficacy of biperiden in preventing the development of epilepsy in patients that suffered TBI, in a double blind, randomized, placebo-controlled trial.

Marmosets have a greater diversity of c-Fos response after hyperstimulation in distinct cortical regions as compared to rats.

Previous evidence indicated a potential mechanism that might support the fact that primates exhibit greater neural integration capacity as a result of the activation of different structures of the central nervous system, as compared to rodents. The current study aimed to provide further evidence to confirm previous findings by analyzing the patterns of c-Fos expression in more neocortical structures of rats and marmosets using a more robust quantitative technique and evaluating a larger number of brain areas. Nineteen Wistar rats and 21 marmosets (Callithrix jacchus) were distributed among control groups (animals without injections) and animals injected with pentylenetetrazol (PTZ) and euthanized at different time points after stimulus. Immunohistochemical detection of c-Fos was quantified using unbiased and efficient stereological cell counting in eight neocortical regions. Marmosets had a c-Fos expression that was notably more widely expressed (5× more cells) and longer lasting (up to 3 hr) than rats. c-Fos expression in rats presented similar patterns of expression according to the function of the brain cortical structures (associative, sensorial, and motor functions), which was not observed for marmosets (in which no clear pattern could be drawn, and a more diverse profile emerged). Our results provide evidence that the marmoset brain has a greater neuronal activation after intense stimulation by means of PTZ and a more complex pattern of brain activation. We speculate that these functional differences may contribute for the understanding of the different neuronal processing capacities of the neocortex in these mammals' orders.

Treatment with CCR2 antagonist is neuroprotective but does not alter epileptogenesis in the pilocarpine rat model of epilepsy.

Neuroinflammation role on epileptogenesis has been the subject of increasing interest. Many studies showed elevation in cytokines and chemokines expression following seizures, such as, CCL2 protein (C-C motif ligand 2 chemokine) and its specific receptor, CCR2. In addition, recent studies manipulating the CCL2/CCR2 complex verified improved seizure outcome in different seizure models. In the present study, the effects of CCR2 antagonist was investigated using the pilocarpine rat model of epilepsy. Status epilepticus (SE) was induced by pilocarpine i.p. injection in adult rats. Daily oral treatment with CCR2 antagonist or vehicle was initiated 5 h following SE and lasted 5 or 10 days. Rats were euthanized 5 days after SE to evaluate neuronal damage and glial density or 30 days after SE to investigate spontaneous seizures development and seizure susceptibility to a second hit pentylenetetrazol (PTZ) test. Rats that received CCR2 antagonist presented less degenerating cells at hippocampal CA1 region. There was also a significant decrease in CA1 volume after SE that was not observed in treated rats. On the other hand, microglia cell density increased after SE regardless of CCR2 antagonist use. Treatment with CCR2 antagonist did not alter spontaneous seizure occurrence or later seizure susceptibility to PTZ in chronic rats. Additional rats were pretreated with CCR2 antagonist prior to SE induction, but this did not change SE progression. The data show that oral treatment with CCR2 antagonist is neuroprotective, but does not alter other epileptogenic factors, such as, neuroinflammation, or seizure development, after pilocarpine-induced SE in rats.

LPS-Induced Systemic Neonatal Inflammation: Blockage of P2X7R by BBG Decreases Mortality on Rat Pups and Oxidative Stress in Hippocampus of Adult Rats.

Neonatal lipopolysaccharide (LPS) exposure-induced brain inflammation has been associated to neuronal injury and facilitates the development of models of neurological disorders in adult rats. The P2X7 receptor (P2X7R) plays a fundamental role in the onset and maintenance of the inflammatory cascade. Brilliant blue G (BBG), a P2X7R antagonist, has been shown to effectively promote neuroinflammatory protection. Here, we have investigated the long-term effects of the neonatal systemic inflammation on hippocampal oxidative stress, anxiety behavior and pain sensitivity in adulthood. We hypothesized that P2X7R blockade is able to modulate the effects of inflammation on these variables. Male and female rat pups received LPS and/or BBG solution intraperitoneally on the 1st, 3rd, 5th and 7th postnatal days. The survival rate and body weight were evaluated during the experimental procedures. The animals were submitted to behavioral tests for anxiety (elevated plus maze, EPM) and nociception (hot-plate and tail-flick) and the oxidative stress was measured by superoxide production in the dentate gyrus of the hippocampus using dihydroethidium (DHE) probe. BBG increased the survival rate in LPS-treated rats. No significant differences were found regarding anxiety behavior and pain sensitivity between the experimental groups. Systemic neonatal inflammation leads to a higher production of superoxide anion in the dentate gyrus of the hippocampus in adulthood and BBG inhibited that effect. Our data suggest that blocking the activation of the P2X7R during neonatal systemic inflammation may have a potential neuroprotective effect in adulthood.

The GCN2 inhibitor IMPACT contributes to diet-induced obesity and body temperature control.

IMPACT, a highly conserved protein, is an inhibitor of the eIF2α kinase GCN2. In mammals, it is preferentially expressed in neurons. Knock-down of IMPACT expression in neuronal cells increases basal GCN2 activation and eIF2α phosphorylation and decreases translation initiation. In the mouse brain, IMPACT is particularly abundant in the hypothalamus. Here we describe that the lack of IMPACT in mice affects hypothalamic functions. Impact-/- mice (Imp-KO) are viable and have no apparent major phenotypic defect. The hypothalamus in these animals shows increased levels of eIF2α phosphorylation, as expected from the described role of IMPACT in inhibiting GCN2 and from its abundance in this brain region. When fed a normal chow, animals lacking IMPACT weight slightly less than wild-type mice. When fed a high-fat diet, Imp-KO animals gain substantially less weight due to lower food intake when compared to wild-type mice. STAT3 signaling was depressed in Imp-KO animals even though leptin levels were identical to the wild-type mice. This finding supports the observation that Imp-KO mice have defective thermoregulation upon fasting. This phenotype was partially dependent on GCN2, whereas the lean phenotype was independent of GCN2. Taken together, our results indicate that IMPACT contributes to GCN2-dependent and -independent mechanisms involved in the regulation of autonomic functions in response to energy availability.

c-Jun expression after cerebral hyperstimulation differs between rats and marmosets.

Immediate early genes (IEGs) are a fundamental element in the way we respond and adapt to a variety of stimuli. We have recently reported that IEG response, as measured by c-Fos expression, is different between rodents and primates. Here, we further extend this analysis by assessing the expression of c-Jun, one of the main complements of c-Fos, under the same stimulation protocol. For this, we investigated the immunohistochemical expression of c-Jun (and compared with that previously shown for c-Fos) after stimulation with pentylenetetrazol in the cingulate gyrus, motor cortex, piriform cortex, inferior temporal cortex, and visual cortex of rats and marmosets (Callithrix jacchus), both male and female. Overall the immunohistochemical expression of c-Jun was more intense but remained elevated for a shorter duration in marmosets as compared to rats. These results are in contrast to what we had previously shown for c-Fos. Furthermore, in terms of the temporal profile, c-Fos and c-Jun expression occurred in a complementary manner in rats-the peak of c-Fos expression coincided with low levels of c-jun expression-and in a superimposed manner in marmosets-the peak of c-Fos expression coincided with the peak of c-Jun expression. Since Fos proteins may form dimers with Jun proteins and together control late gene expressions in the cell nucleus, this different expression profile between primates and rodents may bear meaningful impact for how the nervous system reacts and adapts to stimulation.

THC inhibits the expression of ethanol-induced locomotor sensitization in mice.

The motivational circuit activated by ethanol leads to behavioral changes that recruit the endocannabinoid system (ECS). Case reports and observational studies suggest that the use of Cannabis sp. mitigates problematic ethanol consumption in humans. Here, we verified the effects of the two main phytocannabinoid compounds of Cannabis sp., cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC), in the expression of ethanol-induced locomotor sensitization in mice. Male adult DBA/2 mice were exposed to locomotor sensitization by daily intraperitoneal injections of ethanol (2.5 g/kg) for 12 days; control groups received saline. After the acquisition phase, animals were treated with cannabinoids: CBD (2.5 mg/kg); THC (2.5 mg/kg); CBD + THC (1:1 ratio), or vehicle for 4 days with no access to ethanol during this period. One day after the last cannabinoid injection, all animals were challenged with ethanol (2.0 g/kg) to evaluate the expression of the locomotor sensitization. Mice treated with THC alone or THC + CBD showed reduced expression of locomotor sensitization, compared to the vehicle control group. No effects were observed with CBD treatment alone. Our findings showing that phytocannabinoid treatment prevents the expression of behavioral sensitization in mice provide insight into the potential therapeutic use of phytocannabinoids in alcohol-related problems.

Modification of the natural progression of epileptogenesis by means of biperiden in the pilocarpine model of epilepsy.

Brain injuries are often associated with the later development of epilepsy. Evidence suggests that morphological and functional changes occur in the remaining neural tissue during a silent (or latent) period in which no seizures are expressed. It is believed that this silent (reorganization) period may provide a therapeutic window for modifying the natural history of disease progression. Here we provide evidence that biperiden, a muscarinic anticholinergic agent, is able to alter disease progression in an animal model of epilepsy. We observed that biperiden was capable of slowing the manifestation of the first spontaneous epileptic seizure and effectively reduced the severity and number of recurrent, spontaneous epileptic seizures during the animals' lifespan. Biomolecular (microdialysis) and electrophysiological (extracellular field recordings) studies determined that biperiden was capable of elevating the threshold of hippocampal excitability, thereby making the hippocampal glutamatergic pathways less responsive to stimuli when high concentrations of potassium were used in vivo or in vitro. Notably, there was no hindrance of long-term memory or learning (a potential problem given the amnestic nature of biperiden). We conclude that biperiden has antiepileptogenic potential and may represent an opportunity for the prevention of post-traumatic epilepsy.

Evidence of memory generalization in contextual locomotor sensitization induced by amphetamine.

Addiction is a multifactorial disease that comprises physiological mechanisms of learning and memory. Addict subjects have intense plasticity in cortical and limbic circuits during intoxication, abstinence or even in drug seeking behavior. Locomotor sensitization is a classic animal model of drug addiction that mimics the changes that occur in the transition from drug use to drug addiction. Several studies have demonstrated the importance of contextual associative processes in this task. However, whether the mechanisms of sensitization are maintained and precise over the time remain an open question. Thus, the aim of this study was to investigate the importance of context in the maintenance and precision of locomotor sensitization across time. For this, male c57bl/6 mice were submitted to different contexts during the acquisition phase of amphetamine-induced locomotor sensitization. We found that after 3days of withdrawal, the expression of locomotor sensitization was context dependent, as characterized by an increased locomotion in the acquisition context (A), but not in the novel context (B). Surprisingly, when the expression of locomotor sensitization was tested after 28days of withdrawal, mice that acquired sensitization in the context A exhibited increased locomotion in both contexts (A and B), suggesting that memories associated with amphetamine drugs generalize following long periods of abstinence. The same generalization did not occur in mice sensitized in a well-known context (home cage). These results demonstrate, for the first time, the influence of memory generalization in amphetamine-induced locomotor sensitization. The evidence that memory generalization also occurs in sensitization provides new advances in the comprehension of the mechanisms underlying memory role in addiction process. Elucidating the mechanisms of amphetamine sensitization may shed some light on understanding the transition from drug use to addiction.

Seizures triggered by pentylenetetrazol in marmosets made chronically epileptic with pilocarpine show greater refractoriness to treatment.

The efficiency of most of the new antiepileptic drugs (AEDs) on clinical trials still falls short the success reported in pre-clinical studies, possibly because the validity of the animal models is insufficient to fully represent the human pathology. To improve the translational value for testing AEDs, we propose the use of non-human primates. Here, we suggest that triggering limbic seizures with low doses of PTZ in pilocarpine-treated marmosets might provide a more effective basis for the development of AED. Marmosets with epileptic background were more susceptible to seizures induced by PTZ, which were at least 3 times longer and more severe (about 6 times greater frequency of generalized seizures) in comparison to naïve peers. Accordingly, PTZ-induced seizures were remarkably less attenuated by AEDs in epileptic than naïve marmosets. While phenobarbital (40mg/kg) virtually abolished seizures regardless of the animal's background, carbamazepine (120mg/kg) and valproic acid (400mg/kg) could not prevent PTZ-induced seizures in epileptic animals with the same efficiency as observed in naïve peers. VPA was less effective regarding the duration of individual seizures in epileptic animals, as assessed in ECoG (p=0.05). Similarly following CBZ treatment, the behavioral manifestation of generalized seizures lasted longer in epileptic (p<0.05), which were also more frequent than in the naïve group (p<0.05). As expected, epileptic marmosets experiencing stronger seizures showed more NPY- and ΔFosB-immunostained neurons in a number of brain areas associated with the generation and spread of limbic seizures. Our results suggest that PTZ induced seizures over an already existing epileptic background constitutes a reliable and controllable mean for the screening of new AEDs.

Replacement of Asymmetric Synaptic Profiles in the Molecular Layer of Dentate Gyrus Following Cycloheximide in the Pilocarpine Model in Rats.

Mossy fiber sprouting is among the best-studied forms of post-lesional synaptic plasticity and is regarded by many as contributory to seizures in both humans and animal models of epilepsy. It is not known whether mossy fiber sprouting increases the number of synapses in the molecular layer or merely replaces lost contacts. Using the pilocarpine (Pilo) model of status epilepticus to induce mossy fiber sprouting, and cycloheximide (CHX) to block this sprouting, we evaluated at the ultrastructural level the number and type of asymmetric synaptic contacts in the molecular layer of the dentate gyrus. As expected, whereas Pilo-treated rats had dense silver grain deposits in the inner molecular layer (IML) (reflecting mossy fiber sprouting), pilocarpine + cycloheximide (CHX + Pilo)-treated animals did not differ from controls. Both groups of treated rats (Pilo group and CHX + Pilo group) had reduced density of asymmetric synaptic profiles (putative excitatory synaptic contacts), which was greater for CHX-treated animals. For both treated groups, the loss of excitatory synaptic contacts was even greater in the outer molecular layer than in the best-studied IML (in which mossy fiber sprouting occurs). These results indicate that mossy fiber sprouting tends to replace lost synaptic contacts rather than increase the absolute number of contacts. We speculate that the overall result is more consistent with restored rather than with increased excitability.

The pattern of c-Fos expression and its refractory period in the brain of rats and monkeys.

Intense activation of neurons triggers the appearance of immediate expression genes, including c-Fos. This gene is related to various signal cascades involved in biochemical processes such as neuronal plasticity, cell growth and mitosis. Here we investigate the expression pattern and the refractory period of c-Fos in rats and monkey's brains after stimulation with pentylenetetrazol. Rats and monkeys were sacrificed at various times after PTZ-induced seizure. Here we show that rats and monkeys already showed c-Fos expression at 0.5 h after seizure. Yet, the pattern of protein expression was longer in monkeys than rats, and also was not uniform (relative intensity) across different brain regions in monkeys as opposed to rats. In addition monkeys had a regional brain variation with regard to the temporal profile of c-Fos expression, which was not seen in rats. The refractory period after a second PTZ stimulation was also markedly different between rats and monkeys with the latter even showing a summatory effect on c-Fos expression after a second stimulation. However, assessment of c-Fos mRNA in rats indicated a post-transcriptional control mechanism underlying the duration of the refractory period. The difference in the protein expression pattern in rodents and primates characterizes a functional aspect of brain biochemistry that differs between these mammalian orders and may contribute for the more developed primate cognitive complexity as compared to rodents given c-Fos involvement in cognitive and learning tasks.

Maximal electroshock-induced seizures are able to induce Homer1a mRNA expression but not pentylenetetrazole-induced seizures.

OBJECTIVE: Homer1a is a protein that regulates metabotropic glutamate receptors involved in neural plasticity processes. Recently, we demonstrated that Homer1a mRNA is enhanced after pilocarpine-induced status epilepticus. Here, we investigated whether a single acute seizure triggered by means of pentylenetetrazole (PTZ) injection or maximal electroshock (MES) stimulation (2 different seizure models) would alter Homer1a expression in the hippocampus. METHODS: Male Wistar rats subjected to the PTZ or MES model were analyzed 2h, 8h, 24h, and 7days after seizure induction. Homer1a, mGluR1, and mGluR5 mRNA expression levels in hippocampal extracts were analyzed by quantitative PCR. RESULTS: Quantitative PCR revealed Homer1a overexpression at 2h after MES-induced tonic-clonic seizures compared to control, but the overexpression did not remain elevated after 8h. Pentylenetetrazole-induced seizures, in contrast, were not able to change Homer1a mRNA expression. No differences were observed at these time points after seizures for mGluR1 and mGluR5 mRNA expression in any of the models. SIGNIFICANCE: Our data indicate that the levels of Homer1a mRNA were transiently increased only after MES-induced tonic-clonic seizures (and not after PTZ-induced seizures). We suggest that Homer1a expression may be dependent on seizure intensity or on specific brain circuit activation. We suggest that Homer1a may contribute to counteract hyperexcitability processes.

Role of adenosine in the antiepileptic effects of deep brain stimulation.

Despite the effectiveness of anterior thalamic nucleus (AN) deep brain stimulation (DBS) for the treatment of epilepsy, mechanisms responsible for the antiepileptic effects of this therapy remain elusive. As adenosine modulates neuronal excitability and seizure activity in animal models, we hypothesized that this nucleoside could be one of the substrates involved in the effects of AN DBS. We applied 5 days of stimulation to rats rendered chronically epileptic by pilocarpine injections and recorded epileptiform activity in hippocampal slices. We found that slices from animals given DBS had reduced hippocampal excitability and were less susceptible to develop ictal activity. In live animals, AN DBS significantly increased adenosine levels in the hippocampus as measured by microdialysis. The reduced excitability of DBS in vitro was completely abolished in animals pre-treated with A1 receptor antagonists and was strongly potentiated by A1 receptor agonists. We conclude that some of the antiepileptic effects of DBS may be mediated by adenosine.

Changes in Hippocampal Volume are Correlated with Cell Loss but Not with Seizure Frequency in Two Chronic Models of Temporal Lobe Epilepsy.

Kainic acid (KA) or pilocarpine (PILO) have been used in rats to model human temporal lobe epilepsy (TLE) but the distribution and severity of structural lesions between these two models may differ. Magnetic resonance imaging (MRI) studies have used quantitative measurements of hippocampal T2 (T2HP) relaxation time and volume, but simultaneous comparative results have not been reported yet. The aim of this study was to compare the MRI T2HP and volume with histological data and frequency of seizures in both models. KA- and PILO-treated rats were imaged with a 2 T MRI scanner. T2HP and volume values were correlated with the number of cells, mossy fiber sprouting, and spontaneous recurrent seizures (SRS) frequency over the 9 months following status epilepticus (SE). Compared to controls, KA-treated rats had unaltered T2HP, pronounced reduction in hippocampal volume and concomitant cell reduction in granule cell layer, CA1 and CA3 at 3 months post SE. In contrast, hippocampal volume was unchanged in PILO-treated animals despite detectable increased T2HP and cell loss in granule cell layer, CA1 and CA3. In the following 6 months, MRI hippocampal volume remained stable with increase of T2HP signal in the KA-treated group. The number of CA1 and CA3 cells was smaller than age-matched CTL group. In contrast, PILO group had MRI volumetric reduction accompanied by reduction in the number of CA1 and CA3 cells. In this group, T2HP signal was unaltered at 6 or 9 months after status. Reductions in the number of cells were not progressive in both models. Notably, the SRS frequency was higher in PILO than in the KA model. The volumetry data correlated well with tissue damage in the epileptic brain, suggesting that MRI may be useful for tracking longitudinal hippocampal changes, allowing the assessment of individual variability and disease progression. Our results indicate that the temporal changes in hippocampal morphology are distinct for both models of TLE and that these are not significantly correlated to the frequency of SRS.

Effects of anterior thalamic nucleus deep brain stimulation in chronic epileptic rats.

Deep brain stimulation (DBS) has been investigated for the treatment of epilepsy. In rodents, an increase in the latency for the development of seizures and status epilepticus (SE) has been reported in different animal models but the consequences of delivering stimulation to chronic epileptic animals have not been extensively addressed. We study the effects of anterior thalamic nucleus (AN) stimulation at different current intensities in rats rendered epileptic following pilocarpine (Pilo) administration. Four months after Pilo-induced SE, chronic epileptic rats were bilaterally implanted with AN electrodes or had sham-surgery. Stimulation was delivered for 6 h/day, 5 days/week at 130 Hz, 90 µsec. and either 100 µA or 500 µA. The frequency of spontaneous recurrent seizures in animals receiving stimulation was compared to that recorded in the preoperative period and in rats given sham treatment. To investigate the effects of DBS on hippocampal excitability, brain slices from animals receiving AN DBS or sham surgery were studied with electrophysiology. We found that rats treated with AN DBS at 100 µA had a 52% non-significant reduction in the frequency of seizures as compared to sham-treated controls and 61% less seizures than at baseline. Animals given DBS at 500 µA had 5.1 times more seizures than controls and a 2.8 fold increase in seizure rate as compared to preoperative values. In non-stimulated controls, the average frequency of seizures before and after surgery remained unaltered. In vitro recordings have shown that slices from animals previously given DBS at 100 µA had a longer latency for the development of epileptiform activity, shorter and smaller DC shifts, and a smaller spike amplitude compared to non-stimulated controls. In contrast, a higher spike amplitude was recorded in slices from animals given AN DBS at 500 µA.

Social isolation disrupts hippocampal neurogenesis in young non-human primates.

Social relationships are crucial for the development and maintenance of normal behavior in non-human primates. Animals that are raised in isolation develop abnormal patterns of behavior that persist even when they are later reunited with their parents. In rodents, social isolation is a stressful event and is associated with a decrease in hippocampal neurogenesis but considerably less is known about the effects of social isolation in non-human primates during the transition from adolescence to adulthood. To investigate how social isolation affects young marmosets, these were isolated from other members of the colony for 1 or 3 weeks and evaluated for alterations in their behavior and hippocampal cell proliferation. We found that anxiety-related behaviors like scent-marking and locomotor activity increased after social isolation when compared to baseline levels. In agreement, grooming-an indicative of attenuation of tension-was reduced among isolated marmosets. These results were consistent with increased cortisol levels after 1 and 3 weeks of isolation. After social isolation (1 or 3 weeks), reduced proliferation of neural cells in the subgranular zone of dentate granule cell layer was identified and a smaller proportion of BrdU-positive cells underwent neuronal fate (doublecortin labeling). Our data is consistent with the notion that social deprivation during the transition from adolescence to adulthood leads to stress and produces anxiety-like behaviors that in turn might affect neurogenesis and contribute to the deleterious consequences of prolonged stressful conditions.

Therapeutic effects of the transplantation of VEGF overexpressing bone marrow mesenchymal stem cells in the hippocampus of murine model of Alzheimer's disease.

Alzheimer's disease (AD) is clinically characterized by progressive memory loss, behavioral and learning dysfunction and cognitive deficits, such as alterations in social interactions. The major pathological features of AD are the formation of senile plaques and neurofibrillary tangles together with neuronal and vascular damage. The double transgenic mouse model of AD (2xTg-AD) with the APPswe/PS1dE9 mutations shows characteristics that are similar to those observed in AD patients, including social memory impairment, senile plaque formation and vascular deficits. Mesenchymal stem cells (MSCs), when transplanted into the brain, produce positive effects by reducing amyloid-beta (Aß) deposition in transgenic amyloid precursor protein (APP)/presenilins1 (PS1) mice. Vascular endothelial growth factor (VEGF), exhibits neuroprotective effects against the excitotoxicity implicated in the AD neurodegeneration. The present study investigates the effects of MSCs overexpressing VEGF in hippocampal neovascularization, cognitive dysfunction and senile plaques present in 2xTg-AD transgenic mice. MSC were transfected with vascular endothelial growth factor cloned in uP vector under control of modified CMV promoter (uP-VEGF) vector, by electroporation and expanded at the 14th passage. 2xTg-AD animals at 6, 9 and 12 months old were transplanted with MSC-VEGF or MSC. The animals were tested for behavioral tasks to access locomotion, novelty exploration, learning and memory, and their brains were analyzed by immunohistochemistry (IHC) for vascularization and Aß plaques. MSC-VEGF treatment favored the neovascularization and diminished senile plaques in hippocampal specific layers. Consequently, the treatment was able to provide behavioral benefits and reduce cognitive deficits by recovering the innate interest to novelty and counteracting memory deficits present in these AD transgenic animals. Therefore, this study has important therapeutic implications for the vascular damage in the neurodegeneration promoted by AD.