Maria-Leonor Lopez-Meraz

Low frequency stimulation modifies receptor binding in rat brain

Experiments were designed to reproduce the antiepileptic effects of low frequency stimulation (LFS) during the amygdala kindling process and to examine LFS-induced changes in receptor binding levels of different neurotransmitters in normal brain. Male Wistar rats were stereotactically implanted in the right amygdala with a bipolar electrode. Rats (n = 14) received twice daily LFS (15 min train of 1Hz, 0.1 ms at an intensity of 100 to 400 microA) immediately after amygdala kindling stimulation (1s train of 60 Hz biphasic square waves, each 1 ms at amplitude of 200-500 microA) during 20 days. The LFS suppressed epileptogenesis (full attainment of stage V kindling) but not the presence of partial seizures (lower stages of kindling) in 85.7% of the rats. Thereafter, normal rats (n = 7) received amygdala LFS twice daily for 40 trials. Animals were sacrificed 24 h after last stimulation and their brain used for labeling mu opioid, benzodiazepine (BZD), alpha(1)-adrenergic, and adenylyl cyclase binding. Autoradiography experiments revealed increased BZD receptor binding in basolateral amygdala (20.5%) and thalamus (29.3%) ipsilateral to the place of stimulation and in contralateral temporal cortex (18%) as well as decreased values in ipsilateral frontal cortex (24.2%). Concerning mu receptors, LFS decreased binding values in ipsilateral sensorimotor (7.2%) and temporal (5.6%) cortices, dentate gyrus (5.8% ipsi and 6.8% contralateral, respectively), and contralateral CA1 area of dorsal hippocampus (5.5%). LFS did not modify alpha(1) receptor and adenylyl cyclase binding values. These findings suggest that the antiepileptic effects of LFS may involve activation of GABA-BZD and endogenous opioid systems.

Vulnerability of postnatal hippocampal neurons to seizures varies regionally with their maturational stage.

The mechanism of status epilepticus-induced neuronal death in the immature brain is not fully understood. In the present study, we examined the contribution of caspases in our lithium-pilocarpine model of status epilepticus in 14 days old rat pups. In CA1, upregulation of caspase-8, but not caspase-9, preceded caspase-3 activation in morphologically necrotic cells. Pretreatment with a pan-caspase inhibitor provided neuroprotection, showing that caspase activation was not an epiphenomenon but contributed to neuronal necrosis. By contrast, upregulation of active caspase-9 and caspase-3, but not caspase-8, was detected in apoptotic dentate gyrus neurons, which were immunoreactive for doublecortin and calbindin-negative, two features of immature neurons. These results suggest that, in cells which are aligned in series as parts of the same excitatory hippocampal circuit, the same seizures induce neuronal death through different mechanisms. The regional level of neuronal maturity may be a determining factor in the execution of a specific death program.

5-HT1A receptor agonists modify epileptic seizures in three experimental models in rats

The effects of two serotonergic (5-HT1A) receptor agonists (8-OH-DPAT; 0.01, 0.1, 0.3, 1 mg/kg, s.c., and Indorenate; 1, 3, 10 mg/kg, i.p.) were evaluated in three type of seizures in male Wistar rats: clonic-tonic convulsions induced by pentylenetetrazol (PTZ, 60 mg/kg, i.p.), status epilepticus (SE) of limbic seizures produced by kainic acid (KA, 10 mg/kg, i.p.) and tonic-clonic seizures by amygdala kindling. 8-OH-DPAT decreased the incidence of tonic seizures and the mortality rate induced by PTZ. Indorenate increased the latency to the PTZ-induced seizures and decreased the percentage of rats showing tonic extension and death. Concerning KA, 8-OH-DPAT augmented the latency and reduced the frequency of wet-dog shake (WDS) and generalized seizure (GS). At high doses it diminished the occurrence and delayed the establishment of SE. Indorenate augmented the latency to WDS, GS and SE, and diminished the number of GS. 8-OH-DPAT and Indorenate did not alter the expression of kindled seizures. However, Indorenate enhanced the refractoriness to subsequent seizures during the postictal depression. Some effects induced by 8-OH-DPAT and Indorenate on seizures evaluated and postictal depression were fully or partially blocked by WAY100635. These results suggest that 5-HT1A receptor agonists modify epileptic activity depending on the type of seizure.

Distinct caspase pathways mediate necrosis and apoptosis in subpopulations of hippocampal neurons after status epilepticus

Status epilepticus in the immature brain induces neuronal injury in the hippocampal formation, but the mode and mechanism of death are poorly understood. Our laboratory has recently investigated the role of caspase‐3, ‐8, and ‐9 in neuronal injury, using a lithium–pilocarpine model of status epilepticus in 2‐week‐old rat pups. Our results showed that dying neurons in the dentate gyrus and CA1‐subiculum area do not share the same mechanism of death. In CA1‐subiculum, caspase‐8 upregulation preceded caspase‐3 activation in morphologically necrotic neurons. The pan‐caspase inhibitor Q‐VD‐OPH reduced CA1 damage, showing that caspases contribute to status epilepticus–induced necrosis. In the dentate gyrus, dying neurons were caspase‐9 and ‐3 immunoreactive and morphologically apoptotic. It is not clear why the same seizures cause different types of cell death in neurons that are connected in series along the same hippocampal circuit, but the apoptotic dentate neurons express doublecortin, and do not express calbindin‐D28k, suggesting that their immaturity may be a factor in producing an apoptotic mode of death.

CONTRIBUTION OF A MITOCHONDRIAL PATHWAY TO EXCITOTOXIC NEURONAL NECROSIS

It is traditionally thought that excitotoxic necrosis is a passive mechanism that does not require the activation of a cell death program. In this study, we examined the contribution of the cytochrome c‐dependent mitochondrial death pathway to excitotoxic neuronal necrosis, induced by exposing cultured cortical neurons to 1 mM glutamate for 6 hr and blocked by the NMDA antagonist, dizocilpine. Glutamate treatment induced early cytochrome c release, followed by activation of caspase‐9 and caspase‐3. Preincubation with the caspase‐9 inhibitor z‐LEHD‐fmk, the caspase‐3 inhibitor z‐DEVD‐fmk, or the specific pan‐caspase inhibitor Q‐VD‐oph decreased the percentage of propidium iodide‐positive neurons (52.5% ± 3.1%, 39.4% ± 3.5%, 44.6% ± 3%, respectively, vs. 65% ± 3% in glutamate + vehicle). EM studies showed mitochondrial release of cytochrome c in neurons in the early stages of necrosis and cleaved caspase‐3 immunoreactivity in morphologically necrotic neurons. These results suggest that an active mechanism contributes to the demise of a subpopulation of excitotoxic necrotic neurons.

IL-1β increases necrotic neuronal cell death in the developing rat hippocampus after status epilepticus by activating type I IL-1 receptor (IL-1RI)

Interleukin‐1β (IL‐1β) is associated with seizure‐induced neuronal cell death in the adult brain. The contribution of IL‐1β to neuronal injury induced by status epilepticus (SE) in the immature brain remains unclear. In the present study, we investigated the effects of IL‐1β administration on hippocampal neuronal cell death associated with SE in the immature brain, and the role of the type I receptor of IL‐1β (IL‐1RI). SE was induced with lithium–pilocarpine in 14‐days‐old (P14) rat pups. Six hours after SE onset, pups were i.c.v. injected in the right ventricle with IL‐1β (0, 0.3, 3, 30, or 300 ng), 30 ng of IL‐1RI antagonist (IL‐1Ra) alone, or 30 ng of IL‐1Ra plus 3 ng of IL‐1β. As control groups, pups without seizures were injected with 3 ng of IL‐1β or vehicle. Twenty‐four hours after SE onset, neuronal cell death in the CA1 field of dorsal hippocampus was assessed by hematoxylin–eosin, Fluoro‐Jade B and in vivo propidium iodide (PI) staining; expression of active caspase‐3 (aCas‐3) was also determined, using immunohistochemistry. The concentration–response curve of IL‐1β showed a bell‐shape. Only pups injected with 3 ng of IL‐1β after SE showed a significant increase in the number of cells with eosinophilic cytoplasm and pyknotic nuclei, as well as F‐JB positive cells with respect to the vehicle group. This effect was prevented when IL‐1β was injected with IL‐1Ra. Injection of 3 ng of IL‐1β increased the number of PI‐positive cells in CA1 area after SE. Injection of 3 ng of IL‐1β did not produce hippocampal cell death in rats without seizures. Active caspase‐3 expression was not observed after treatments in hippocampus. The activation of the IL‐1β/IL‐1RI system increases necrotic neuronal cell death caused by SE in rat pups.

Do single seizures cause neuronal death in the human hippocampus

The question of whether repeated single seizures cause neuronal death in the adult human brain is of great clinical importance and might have broad therapeutic implications. Reviewed here are recent studies on the effects of repeated single seizures (in the absence of status epilepticus) on hippocampal volume and on neuronal death markers in blood and in surgically ablated hippocampi.

Hyperthermia aggravates status epilepticus-induced epileptogenesis and neuronal loss in immature rats

This study tightly controlled seizure duration and severity during status epilepticus (SE) in postnatal day 10 (P10) rats, in order to isolate hyperthermia as the main variable and to study its consequences. Body temperature was maintained at 39 ± 1 °C in hyperthermic SE rats (HT+SE) or at 35 ± 1 °C in normothermic SE animals (NT+SE) during 30 min of SE, which was induced by lithium-pilocarpine (3 mEq/kg, 60 mg/kg) and terminated by diazepam and cooling to NT. All video/EEG measures of SE severity were similar between HT+SE and NT+SE pups. At 24h, neuronal injury was present in the amygdala in the HT+SE group only, and was far more severe in the hippocampus in HT+SE than NT+SE pups. Separate groups of animals were monitored four months later for spontaneous recurrent seizures (SRS). Only HT+SE animals developed convulsive SRS. Both HT+SE and NT+SE animals developed electrographic SRS (83% vs. 55%), but SRS frequency and severity were higher in hyperthermic animals (12.5 ± 3.5 vs. 4.2 ± 2.0 SRS/day). The density of hilar neurons was lower, thickness of the amygdala and perirhinal cortex was reduced, and lateral ventricles were enlarged in HT+SE over NT+SE littermates and HT/NT controls. In this model, hyperthermia greatly increased the epileptogenicity of SE and its neuropathological sequelae.

Tickling in juvenile but not adult female rats conditions sexual partner preference

Female rats display a conditioned partner preference for males that bear odors paired with different types of rewarding unconditioned stimuli (UCS). Here we examined whether tickling constitutes a rewarding UCS that supports the development of partner preferences. In Experiment 1, we tested the possibility that odors associated with a tickling UCS in prepubescent rats would induce a conditioned partner preference in adulthood. Two groups were formed with 31-day-old, single-housed females, tickled for 6 min daily for 10 days, by a hand that wore a scented glove (almond or lemon). At 47 days of age, females were ovariectomized (OVX), hormone-primed (EB+P), and tested for sexual partner preference with two scented stud males (one almond and one lemon). In each group, females displayed a sexual preference toward males bearing the odor paired with tickling, as observed with longer visits, more solicitations, hops & darts, and receiving more intromissions and ejaculations from the preferred male. In Experiment 2, we used 3-month old, OVX, hormone-primed rats conditioned every 4 days for 10 trials. In contrast to juvenile females, adult females failed to prefer males that bore the odor paired with tickling but instead preferred the novel male. These results suggest that tickling has opposite age-dependent effects in the conditioning of partner preference. Tickling in juvenile females appears to act as a rewarding UCS, whereas in adult females it may act as an aversive UCS. Further research is needed to understand brain mechanisms that might account for such differences.

BEYOND THE BASAL GANGLIA: cFOS EXPRESSION IN THE CEREBELLUM IN RESPONSE TO ACUTE AND CHRONIC DOPAMINERGIC ALTERATIONS

The suggestion of an anatomical and functional relationship between the basal ganglia and cerebellum is recent. Traditionally, these structures were considered as neuronal circuits working separately to organize and control goal-directed movements and cognitive functions. However, several studies in rodents and primates have described an anatomical interaction between cortico-basal and cortico-cerebellar networks. Most importantly, functional changes have been observed in one of these circuits when altering the other one. In this context, we aimed to accomplish an extensive description of cerebellar activation patterns using cFOS expression (cFOS-IR) after acute and chronic manipulation of dopaminergic activity. In the acute study, substantia nigra pars compacta (SNc) activity was stimulated or suppressed by intra cerebral administration of picrotoxin or lidocaine, respectively. In addition, we analyzed cerebellar activity after the induction of a parkinsonism model, the tremulous jaw movements. In this model, tremulous jaw movements were induced in male rats by IP chronic administration of the dopamine antagonist haloperidol (1.5mg/kg). Acute stimulation of SNc by picrotoxin increased cFOS-IR in the vermis and cerebellar hemispheres. However, lidocaine did not produce an effect. After 14days of haloperidol treatment, the vermis and cerebellar hemispheres showed an opposite regulation of cFOS expression. Chronic dopaminergic antagonism lessened cFOS expression in the vermis but up-regulated such expression in the cerebellar hemisphere. Overall, the present data indicate a very close functional relationship between the basal ganglia and the cerebellum and they may allow a better understanding of disorders in which there are dopamine alterations.