The Effects of Optogenetic Activation of Astrocytes on Spike-and-Wave Discharges in Genetic Absence Epileptic Rats.

Background: Absence seizures (petit mal seizures) are characterized by a brief loss of consciousness without loss of postural tone. The disease is diagnosed by an electroencephalogram (EEG) showing spike-wave discharges (SWD) caused by hypersynchronous thalamocortical (TC) oscillations. There has been an explosion of research highlighting the role of astrocytes in supporting and modulating neuronal activity. Despite established in vitro evidence, astrocytes' influence on the TC network remains to be elucidated in vivo in the absence epilepsy (AE). Purpose: In this study, we investigated the role of astrocytes in the generation and modulation of SWDs. We hypothesize that disturbances in astrocytes' function may affect the pathomechanism of AE. Methods: To direct the expression of channelrhodopsin-2 (ChR2) rAAV8-GFAP-ChR2(H134R)-EYFP or to control the effect of surgical intervention, AAV-CaMKIIa-EYFP was injected into the ventrobasal nucleus (VB) of the thalamus of 18 animals. After four weeks following the injection, rats were stimulated using blue light (~473 nm) and, simultaneously, the electrophysiological activity of the frontal cortical neurons was recorded for three consecutive days. The animals were then perfused, and the brain tissue was analyzed by confocal microscopy. Results: A significant increase in the duration of SWD without affecting the number of SWD in genetic absence epileptic rats from Strasbourg (GAERS) compared to control injections was observed. The duration of the SWD was increased from 12.50 ± 4.41 s to 17.44 ± 6.07 following optogenetic stimulation in GAERS. The excitation of the astrocytes in Wistar Albino Glaxo Rijswijk (WAG-Rij) did not change the duration of SWD; however, stimulation resulted in a significant increase in the number of SWD from 18.52 ± 11.46 bursts/30 min to 30.17 ± 18.43 bursts/30 min. Whereas in control injection, the duration and the number of SWDs were similar at pre- and poststimulus. Both the background and poststimulus average firing rates of the SWD in WAG-Rij were significantly higher than the firing recorded in GAERS. Conclusion: These findings suggest that VB astrocytes play a role in modulating the SWD generation in both rat models with distinct mechanisms and can present an essential target for the possible therapeutic approach for AE.

Impact of multiple firings and resin cement type on shear bond strength between zirconia and resin cements.

PURPOSE: The aim of this study was to evaluate the effect of multiple firings on the bond strength between yttria-tetragonal zirconia polycrystals (Y-TZP) and 2 types of resin cements. MATERIALS AND METHODS: Sixty 3Y-TZP specimens (LAVA Frame Multi) were divided into 3 groups depending on the following firing procedures: (1) 2-firing cycles, (2) 5-firing cycles, (3) 10-firing cycles. Two samples from each group were investigated by using SEM to determine the morphological changes. All specimens were treated with 125 µm airborne-particle abrasion and the surface roughness of each specimen was measured. The specimens from each firing group were then further divided into 2 subgroups (n = 9) to apply 2 types of resin cement (MDP-free resin cement: RelyX Unicem-RU, and MDP containing resin cement: Panavia F 2.0-PA). The shear bond strength (SBS) test was performed and failure types of all the debonded specimens were classified by using a stereomicroscope as adhesive, cohesive, and mixed. The statistical analysis of surface roughness and SBS data were performed by using 1-way ANOVA and 2-way ANOVA followed by Tukey-HSD tests (α=.05). Failure modes were calculated as a percentage for each group. RESULTS: The bond strength of RU and PA to the specimens obtained with 2 firings were not statistically different from each other (P=.1). However, the SBS values of PA were found to be significantly higher than RU for the specimens obtained with 5 and 10 firing cycles (P=.001 and P=.02, respectively). Surface roughness analysis revealed no statistical difference between groups (P=.2). The SEM analysis of samples fired 5- and 10- times showed irregularities and boundary loss in zirconia grains, and empty spaces between zirconia grains. CONCLUSION: The bond strength of PA cement was higher than that of RU to the zirconia subjected to repeated firings (5 and 10 firing cycles). When zirconia is subjected to multiple firings, using MDP-containing resin cement can be recommended.

Relationships between astrocytes and absence epilepsy in rat: An experimental study.

Astrocytes take part in the modulation of neuronal activity through the uptake and release of both GABA and glutamate. In the present study we aimed to quantify the number of astrocytes expressing the astrocyte marker glial fibrillary acidic protein (GFAP) in the somatosensory cortex (SSCx), ventrobasal (VB), centromedial (CM), reticular (TRN) and dorsal lateral geniculate (dLGN) nuclei of thalamus in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), Wistar Albino Glaxo Rats from Rijswijk (WAG/Rij) and control Wistar animals. Further, we aimed to compare the GFAP protein expression levels between the three animal strains. The GFAP-immunohistochemistry was applied to sections from the SSCx, VB, CM, TRN and dLGN and GFAP-positive astrocytes were quantified for the three animal strains. Further, GFAP Western Blot was applied to the tissue samples from the same regions of the three strain. The data obtained from Wistar animals were compared with GAERS and WAG/Rij animals. The number of GFAP-positive astrocytes per unit area in all brain regions studied showed high significance between Wistar-GAERS and Wistar-WAG/Rij except the dLGN. The GAERS had significant higher endogenous GFAP expression in all brain regions studied compared to Wistar and WAG/Rij animals. These findings demonstrate a discrete difference in both GFAP-positive astrocyte populations and GFAP protein expression levels between Wistar and genetically epileptic strains (GAERS and WAG/Rij). Absence seizures are thought to result from a possible imbalance in glutamatergic and GABAergic neurotransmission. Astrocytes regulate the concentration of glutamate and GABA in the extracellular space in the brain, the difference in the astrocyte population and GFAP protein expression in the epileptic strains clearly shows the involvement of astrocytes in the mechanism of absence epilepsy.

The effect of prenatal and postnatal caffeine exposure on pentylentetrazole induced seizures in the non-epileptic and epileptic offsprings.

Caffeine, a central nervous system stimulant, has been reported to modulate seizure activity in various studies. In this study the effects of caffeine exposure on the pentylenetetrazole (PTZ) induced seizure thresholds and seizure stages in the Wistar and genetic absence epilepsy model offsprings were examined. Adult female and male Wistar rats and genetic absence epilepsy rats from Strasbourg (GAERS) consumed caffeine dissolved in water (0.3 g/L) before conception, during the gestational periods and lactation period whereas control groups of each strain received tap water. All offsprings at postnatal day 30 (PN30) subjected to 70 mg/kg of PTZ were evaluated in terms of overall seizure stages, the latency to the first generalized seizure and the c-Fos protein activity in the brain regions of somatosensorial cortex (SSCx), reticular thalamic nucleus (Rt), ventrobasal thalamus (VB), centromedial nucleus (CM) and lateral geniculate nucleus (LGN). The Wistar caffeine group had significantly shorter latency to the first generalized seizure (1.53 ±â€¯0.49 min) comparing to the Wistar control offsprings (3.40 ±â€¯0.68 min). GAERS caffeine group (6.52 ±â€¯2.48 min) showed significantly longer latency comparing to Wistar caffeine group (1.53 ±â€¯0.49 min). Although statistically not significant, GAERS caffeine group showed a longer latency comparing to the GAERS control group (4.71 ±â€¯1.82 min). In all regions of SSCx, Rt, VB, CM and LGN, GAERS caffeine group had lower c-Fos protein expression comparing to the GAERS control group (p < 0.05). Wistar caffeine rats had lower expression of c-Fos protein comparing to the Wistar control group only in SSCx. In CM, GAERS rats expressed lower c-Fos protein comparing to the Wistar control (p < 0.05). In conclusion differential effects of caffeine in the seizure modulation may involve c-Fos protein activity-dependent protection mechanisms.

Afferent projections of the subthalamic nucleus in the rat: emphasis on bilateral and interhemispheric connections.

The subthalamic nucleus (STN) is important for normal movement as well as in movement disorders. The STN is a target nuclei in patients with advanced Parkinson's disease (PD). Deep brain stimulation (DBS) is a standard surgical treatment for PD. Although DBS results in a significant reduction in motor disability, several negative side effects have been reported. Thus, to understand the side effects of DBS the connection of the STN should be well known. Therefore, the present study aims to re­examine the STN with an emphasis on poorly­ or un­documented connections. Furthermore, the bilateral and interhemispheric connections of the STN are evaluated. Fifteen male albino rats received injections of Fluoro­Gold retrograde and biotinylated dextran amine anterograde tracers into the STN. Following a 7-10 day survival period, the animals were processed according to the relevant protocol for each tracer. The present study demonstrates ipsilateral connections of the STN with cortical regions (i.e., infralimbic, cingulate, frontal, piriform, primary motor, primary sensory, insular and retrosplenial cortices), the endopiriform nucleus, basal ganglia related structures (i.e., caudate putamen, globus pallidus, ventral pallidum, nucleus accumbens, claustrum and substantia innominata) and the deep cerebellar nuclei (i.e., lateral, anterior interposed). Bilateral connections of the STN were observed with limbic (amygdala, bed nucleus of stria terminalis), hypothalamic (ventromedial, posterior, anterior, lateral and mammillary) thalamic (thalamic reticular nucleus), epithalamic (habenular nucleus), and brainstem structures (superior colliculus, substantia nigra, spinal nucleus of the trigeminal nerve, red nucleus, dorsal raphe nucleus, pedunculopontine tegmental nuclei). Interhemispheric connections between left and right STN were also observed. The present study fills important gaps in connectivity of the STN. In particular, we report STN connectivity with cortical areas (i.e., piriform, endopiriform and insular), claustrum, hypothalamic, thalamic reticular, cerebellar, habenular, trigeminal, red, cuneate and gracile nuclei and substantia innominate. These connections, which have not been previously described or poorly described, provide new routes that can alter the conceptual architecture of the basal ganglia circuitry and may modify our view of the functional identity of the STN.

Cortical, subcortical and brain stem connections of the cerebellum via the superior and middle cerebellar peduncle in the rat.

The role of cerebellum in coordination of somatic motor activity has been studied in detailed in various species. However, experimental and clinical studies have shown the involvement of the cerebellum with various visceral and cognitive functions via its vast connections with the central nervous system. The present study aims to define the cortical and subcortical and brain stem connections of the cerebellum via the superior (SCP) and middle (MCP) cerebellar peduncle using biotinylated dextran amine (BDA) and Fluoro-Gold (FG) tracer in Wistar albino rats. 14 male albino rats received 20-50-nl pressure injections of either FG or BDA tracer into the SCP and MCP. Following 7-10 days of survival period, the animals were processed according to the related protocol for two tracers. Labelled cells and axons were documented using light and fluorescence microscope. The SCP connects cerebellum to the insular and infralimbic cortices whereas, MCP addition to the insular cortex, it also connects cerebellum to the rhinal, primary sensory, piriform and auditory cortices. Both SCP and MCP connected the cerebellum to the ventral, lateral, posterior and central, thalamic nuclei. Additionally, SCP also connects parafasicular thalamic nucleus to the cerebellum. The SCP connects cerebellum to basal ganglia (ventral pallidum and clastrum) and limbic structures (amygdaloidal nuclei and bed nucleus of stria terminalis), however, the MCP have no connections with basal ganglia or limbic structures. Both the SCP and MCP densely connects cerebellum to various brainstem structures. Attaining the knowledge of the connections of the SCP and MCP is important for the diagnosis of lesions in the MCP and SCP and would deepen current understanding of the neuronal circuit of various diseases or lesions involving the SCP and MCP.

Assessment of the corticospinal fiber integrity in mirror movement disorder.

Mirror movements are unintended movements occurring on one side of the body that mirror the contralateral voluntary ones. It has been proposed that mirror movements occur due to abnormal decussation of the corticospinal pathways. Using detailed multidisciplinary approach, we aimed to enlighten the detailed mechanism underlying the mirror movements in a case subject who is diagnosed with mirror movements of the hands and we compared the findings with the unaffected control subjects. To evaluate the characteristics of mirror movements, we used several techniques including whole exome sequencing, computed tomography, diffusion tensor imaging and transcranial magnetic stimulation. Computed tomography showed the absence of a spinous process of C5, fusion of the body of C5-C6 vertebrae, hypoplastic dens and platybasia of the posterior cranial fossa. A syrinx cavity was present between levels C3-C4 of the spinal cord. Diffusion tensor imaging of the corticospinal fibers showed disorganization and minimal decussations at the lower medulla oblongata. Transcranial magnetic stimulation showed that motor commands were distributed to the motor neuron pools on the left and right sides of the spinal cord via fast-conducting corticospinal tract fibers. Moreover, a heterozygous missense variation in the deleted in colorectal carcinoma gene has been observed. Developmental absence of the axonal guidance molecules or their receptors may result in abnormalities in the leading of the corticospinal fibers. Clinical evaluations and basic neuroscience techniques, in this case, provide information for this rare disease and contribute to our understanding of the normal physiology of bimanual coordination.

The Cerebello-Hypothalamic and Hypothalamo-Cerebellar Pathways via Superior and Middle Cerebellar Peduncle in the Rat.

The connections between the cerebellum and the hypothalamus have been well documented. However, the specific cerebellar peduncle through which the hypothalamo-cerebellar and cerebello-hypothalamic connections pass has not been demonstrated. The present study aims to define the specific cerebellar peduncle through which connects the cerebellum to specific hypothalamic nuclei. Seventeen male albino rats received 20-50-nl pressure injections of either Fluoro-Gold (FG) or biotinylated dextran amine (BDA) tracer into the superior (SCP), middle (MCP), and inferior (ICP) cerebellar peduncle. Following 7-10 days of survival period, the animals were processed according to the appropriate protocol for the two tracers used. Labeled cells and axons were documented using light or fluorescence microscopy. The present study showed connections between the hypothalamus and the cerebellum via both the SCP and the MCP but not the ICP. The hypothalamo-cerebellar connections via the SCP were from the lateral, dorsomedial, paraventricular, and posterior hypothalamic nuclei, and cerebello-hypothalamic connections were to the preoptic and lateral hypothalamic nuclei. The hypothalamo-cerebellar connections via the MCP were from the lateral, dorsomedial, ventromedial, and mammillary hypothalamic nuclei; and cerebello-hypothalamic connections were to the posterior, arcuate, and ventromedial hypothalamic nuclei. The hypothlamo-cerebellar connections were denser compared to the cerebello-hypothlamic connections via both the SCP and the MCP. The connection between the cerebellum and the hypothalamus was more prominent via the SCP than MCP. Both the hypothlamo-cerebellar and cerebello-hypothalamic connections were bilateral, with ipsilateral preponderance. Reciprocal connections were with the lateral hypothalamic nucleus via the SCP and the ventromedial nucleus via the MCP were observed. Cerebellum takes part in the higher order brain functions via its extensive connections. The knowledge of hypothalamo-cerebellar and cerebello-hypothalamic connections conveyed within the SCP and MCP can be important for the lesions involving the MCP and SCP. These connections can also change the conceptual architecture of the cerebellar circuitry and deepen current understanding.

Comparing glutamatergic neuron population in the mediodorsal thalamic nucleus of genetic absence epilepsy rats from strasbourg (GAERS) and normal control Wistar rats.

An imbalance between GABAergic inhibition and glutamatergic excitation is suspected to play a role in the genesis of epileptic processes. In the present study we quantified the number of glutamate+ve neurons in the mediodorsal thalamic nucleus (MD) of genetic absence epilepsy rats from Strasbourg (GAERS) and compared these with values for normal Wistar rats. The MD thalamic nucleus was removed from each animal and the glutamatergic neurons were labelled using light-microscopy glutamate immunohistochemistry. The disector method was used to quantify the glutamate+ve neurons in the MD thalamic nucleus of GAERS and Wistar rats. The data were statistically analyzed. In the Wistar animals glutamate+ve neurons formed 89% and in GAERS 92.3% of the total neurons in 1000µm3 of MD thalamic nucleus. In GAERS glutamate+ve neurons showed statistically significant increase in the MD thalamic nucleus compared to Wistar animals. In Wistar animals the glutamate-ve neurons formed 11% and in GAERS 7.7% of the total neurons in 1000µm3 of MD thalamic. No significant difference was observed in glutamate-ve neurons between the two strains. The average diameter of glutamate+ve neurons showed no significance, while glutamate-ve neurons were significant between the two strains. The results of the present study, on genetic absence epilepsy model, GAERS, confirms the role of MD thalamic nucleus in chemically induced absence epilepsy.

Motor afferents from the cerebellum, zona incerta and substantia nigra to the mediodorsal thalamic nucleus in the rat.

The mediodorsal (MD) thalamic nucleus provides information from subcortical structures to the prefrontal cortex. The human MD thalamic nucleus has been implicated in a great variety of different clinical conditions and normal functions ranging from schizophrenia, Parkinsonism and epilepsy to many cognitive functions. In the rat the MD thalamic nucleus is divided into three cytoarchitectonic sectors whereas in the primates it is divided into two; medial one-third (magnocellular) and lateral two-thirds further the lateral sector is divided into pars parvocellularis pars multiformis, pars fasciculosa and pars caudalis. In this study we used a retrograde tracer, fluoro-gold (FG) to evaluate some of the afferents reaching the lateral sector of the MD (MDl) thalamic nucleus. The results of the present study have shown that MDl receives afferent connections from the lateral cerebellar nucleus (dentate nucleus), substantia nigra pars reticulata (SNR) and zona incerta (ZI). Subsequent to FG injections into the MDl, labeled cells were observed mainly bilaterally but were sparser on the contralateral side than ipsilaterally from each of the three structures listed. All three afferents showed a topographical organization. The labeled neurons were localized at the dorsomedial aspect of the lateral cerebellar nucleus, the dorsoventral aspect of the SNR and in the dorsal sector of the ZI. The lateral cerebellar nucleus reached the MDl via the superior cerebellar peduncle. No other deep cerebellar nuclei showed labeled cells. There were no labeled cells in the substantia nigra pars compacta (SNC). Although the three regions identified here are recognized as having motor functions, the connections to MD suggest that their outputs also play a role in cognitive or other higher cortical functions.

Differential dopamine receptor occupancy underlies L-DOPA-induced dyskinesia in a rat model of Parkinson's disease.

Dyskinesia is a major side effect of an otherwise effective L-DOPA treatment in Parkinson's patients. The prevailing view for the underlying presynaptic mechanism of L-DOPA-induced dyskinesia (LID) suggests that surges in dopamine (DA) via uncontrolled release from serotonergic terminals results in abnormally high level of extracellular striatal dopamine. Here we used high-sensitivity online microdialysis and PET imaging techniques to directly investigate DA release properties from serotonergic terminals both in the parkinsonian striatum and after neuronal transplantation in 6-OHDA lesioned rats. Although L-DOPA administration resulted in a drift in extracellular DA levels, we found no evidence for abnormally high striatal DA release from serotonin neurons. The extracellular concentration of DA remained at or below levels detected in the intact striatum. Instead, our results showed that an inefficient release pool of DA associated with low D2 receptor binding remained unchanged. Taken together, these findings suggest that differential DA receptor activation rather than excessive release could be the underlying mechanism explaining LID seen in this model. Our data have important implications for development of drugs targeting the serotonergic system to reduce DA release to manage dyskinesia in patients with Parkinson's disease.