Lack of starvation-induced activation of AMP-activated protein kinase in the hypothalamus of the Lou/C rats resistant to obesity.

OBJECTIVE: The AMP-activated protein kinase (AMPK) is involved in the control of food intake by the hypothalamus. The aim of this work was to investigate if modification of hypothalamic AMPK regulation could be related to the spontaneous food restriction of Lou/C rats, a strain resistant to obesity exhibiting a 40% reduction in caloric intake compared with their lean Wistar counterparts. DESIGN: Three-month-old male Lou/C rats were compared with age-matched male Wistar rats in both fed ad libitum and 24-h food deprivation state. MEASUREMENTS AND RESULTS: We first confirmed that starvation activated both isoforms of AMPK catalytic alpha subunits and enhanced the phosphorylation state of its downstream targets acetyl-CoA carboxylase and elongation factor 2 in the hypothalamus of Wistar rats. These changes were not observed in the hypothalamus of Lou/C rats. Interestingly, the starvation-induced changes in hypothalamic mRNA levels of the main orexigenic and anorexigenic neuropeptides were also blunted in the Lou/C rats. Analysis of the concentrations of circulating substrates and hormones known to regulate hypothalamic AMPK indicated that the starvation-induced changes in ghrelin, adiponectin and leptin were not observed in Lou/C rats. Furthermore, an increased phosphorylation state of signal transducer and activator of transcription 3 (STAT3), which admittedly mediates leptin signaling, was evidenced in the hypothalamus of the starved Lou/C rats, as well as modifications of expression of the leptin-sensitive genes suppressor of cytokine signaling-3 and stearoyl-coenzyme A desaturase 1. In addition, despite reduced leptin level in fed Lou/C rats, the phosphorylation state of hypothalamic STAT3 remained similar to that found in fed Wistar rats, an adaptation that could be explained by the concomitant increase in ObRb leptin receptor mRNA expression. CONCLUSION: Activation of hypothalamic AMPK by starvation, which stimulates food intake through changes in (an)orexigenic neuropeptides in the normal rats, was not observed in the spontaneously hypophagic Lou/C rats.

Up-regulation of D3 dopaminergic receptor mRNA in the core of the nucleus accumbens accompanies the development of seizures in a genetic model of absence-epilepsy in the rat.

The basal ganglia system is thought to play a key role in the control of absence-seizures and there is ample evidence that epileptic seizures modify brain dopamine function. We recently reported that local injections of dopamine D1 or D2 agonists in the core of the nucleus accumbens suppressed absence-seizures in a spontaneous, genetic rodent model of absence-epilepsy whereas injections of D1 or D2 antagonists had aggravating effects. These findings raised the possibility that the dopaminergic system may be altered in absence-epilepsy prone rats. Therefore, we studied by in situ hybridization histochemistry the expression of pre- and postsynaptic components of the dopaminergic system in this strain of rats. When compared to non-epileptic control rats, epileptic rats displayed no change in the expression of mRNAs coding for the neuronal dopaminergic markers (tyrosine hydroxylase, membraneous and vesicular dopamine transporters). In addition, there was no difference between the two strains concerning the expression of the dopamine receptor transcripts D1, D2 and D5. In adult absence-epilepsy prone rat with an overt epileptic phenotype, however, an elevated level of D3 mRNA expression was observed in neurons of the core of the nucleus accumbens (+23% increase in silver grain density compared to non-epileptic control rats). D3 transcripts were not increased in juvenile epileptic rats without seizures. These findings suggests that up-regulation of D3 receptor mRNA is part of the epileptic phenotype in absence-epilepsy prone rats. Its localization in the core of the nucleus accumbens bears close resemblance to the dopamine-sensitive antiepileptic sites in ventral striatum and further support the involvement of ventral structures of the basal ganglia system in the control of absence-seizures.

Inhibition of the substantia nigra suppresses absences and clonic seizures in audiogenic rats, but not tonic seizures: evidence for seizure specificity of the nigral control.

GABAergic inhibition of the substantia nigra pars reticulata has been shown to suppress seizures in most models of epilepsy involving forebrain networks, such as absences or clonic seizures. No such antiepileptic effects were observed, however, in genetically audiogenic rats exhibiting tonic seizures generated in the brainstem. This suggests a constitutive dysfunction of the nigral GABAergic neurotransmission in this strain of rat or a selective action of the nigral control on specific networks. In the present study, we first confirmed that bilateral injection of muscimol (700 pmol/side) in the substantia nigra had no effect in Wistar rats with audiogenic seizures (Wistar AS). [3H]Muscimol autoradiography suggested a 40% reduced density of GABA(A) receptors in the substantia nigra of Wistar AS, whereas no change was observed in the cortex and the superior colliculus (superficial and intermediate layers), as compared to control animals. In Wistar AS where 40 repetitions of audiogenic stimulations progressively induced generalised convulsive seizures with both tonic and clonic components, bilateral injection of muscimol (350 pmol/side) in the substantia nigra suppressed the clonic component but had no effect on tonic seizures. In hybrid rats issued from cross-breeding between Wistar AS and rats with spontaneous absence seizures, bilateral injection of muscimol (18 pmol/side) in the substantia nigra abolished cortical spike-and-wave discharges, but had no effect on tonic audiogenic seizures at doses up to 700 pmol/side. These results show that despite a decreased number of GABA(A) receptors in the substantia nigra, inhibition of this structure in Wistar AS still leads to inhibition of seizures involving forebrain structures. These results confirm that GABAergic inhibition of the substantia nigra has antiepileptic effects through the control of forebrain circuits. They suggest that this control mechanism has no inhibitory effect on circuits underlying audiogenic tonic seizures.

[3H]acetycholine release in rat striatal slices is not subject to dopamine heteroreceptor supersensitivity 30 months after 6-hydroxydopamine lesion of the substantia nigra.

Using the rat model of Parkinson's disease described by Ungerstedt the release of [3H]acetylcholine ([3H]ACh) in the caudatoputamen was investigated to assess possible long-term effects of unilateral dopaminergic denervation on the modulation of cholinergic interneurons. This seemed of interest since rats with 6-hydroxydopamine (6-OHDA) lesions of the left substantia nigra showed an increase in the behavioural susceptibility to small doses of dopamine (DA) D2 receptor agonists 30 months after the lesion. Electrical field stimulation with 3 Hz elicited release of [3H]ACh in slices of both the lesioned and the intact striatum. The DA reuptake blocker nomifensine was ineffective on the lesioned side but diminished the release of [3H]ACh in the intact striatum. This inhibition was reversed by the D2 receptor antagonist domperidone and hence probably due to the effect of endogenously released DA. Single electrical pulses at 0.05 Hz, which neither induced autoinhibition of [3H]ACh release nor heteroinhibition by endogenous DA, elicited a higher release of [3H]ACh on the intact side. Under this stimulation paradigm activation of the D2 heteroreceptor with quinpirole depressed the release of [3H]ACh to a similar extent on both sides, irrespective of the absence or presence of the competitive NMDA receptor antagonist D-CPPene. Also blockade of the NMDA receptor channel by dizocilpine, or of AMPA receptors by NBQX, was ineffective on either side. The NMDA-evoked release of [3H]ACh was higher on the lesioned side. It was equally depressed by quinpirole and by ethanol on both sides. Thus, single electrical pulses and NMDA stimulation per se had opposite effects on the lesioned and the intact side, whereas the modulation of release was similar. Since the lesioned striata were considerably smaller, measurements of mRNA levels of choline acetyltransferase (ChAT) were used to assess the density of cholinergic interneurons and their content of ChAT mRNA. This analysis did not reveal any side difference. In conclusion, the function of D2 heteroreceptors on, and the density and ChAT mRNA content of, cholinergic interneurons are not or no longer altered after long-term DA denervation. Most probably, cholinergic interneurons are not involved in the increased behavioural susceptibility of 6-OHDA-lesioned rats to DA agonists.

Dopamine in the striatum modulates seizures in a genetic model of absence epilepsy in the rat.

Inhibition of the substantia nigra pars reticulata has been shown to suppress seizures in different animal models of epilepsy. The striatum is the main input of the substantia nigra pars reticulata. The aim of the present study was to examine the role of dopaminergic neurotransmission within the striatum in the control of absence seizures in a genetic model in the rat. Injections of mixed dopaminergic D1/D2 or of selective D1 or D2 agonists or antagonists in the dorsal parts of the striatum led to suppression of absence seizures associated with strong behavioral and electroencephalographic side-effects. When injected in the ventral part of the striatum (i.e. the nucleus accumbens core), all these agonists and antagonists respectively decreased and increased absence seizures without behavioral or electroencephalographic side-effects. Combined injections of low doses of a D1 and a D2 agonist in the core of the nucleus accumbens had an additive effect in absence seizures suppression. Furthermore, combined injections of low doses of a GABA(A) agonist and a N-methyl-D-aspartate antagonist in the substantia nigra also had cumulative effects in absence seizures suppression. These results show that dopamine neurotransmission in the core of the nucleus accumbens is critical in the control of absence seizures. The modulatory and additive effects on absence seizures of dopaminergic neurotransmission through both the D1 and D2 receptors in the core of the nucleus accumbens further suggest that ventral pathways of the basal ganglia system are involved in the modulation of absence seizures.

Evidence for the involvement of the pallidum in the modulation of seizures in a genetic model of absence epilepsy in the rat.

Inhibition of the subthalamic nucleus (STN) has been shown to suppress seizures in different animal models of epilepsy. The aim of this study was to examine the role of the pallidal inputs to the STN in the control of absence seizures in a genetic model in the rat. Disinhibition of the globus pallidus or the ventral pallidum, by local injections of a GABA(A) antagonist, suppressed absence seizures. Conversely, inhibition of the ventral pallidum by a GABA(A) agonist aggravated absence seizures. Furthermore, the antiepileptic effects of intrapallidal injections of a GABA(A) antagonist were correlated with a decrease of extracellular levels of glutamate in the substantia nigra. Our results show that both the globus pallidus and the ventral pallidum exert a modulatory influence on absence seizures and suggest that these effects are mediated through the STN.

The role of basal ganglia in the control of generalized absence seizures.

During the last two decades, evidence has accumulated to demonstrate the existence, in the central nervous system, of an endogenous mechanism that exerts an inhibitory control over different forms of epileptic seizures. The substantia nigra and the superior colliculus have been described as key structures in this control circuit; inhibition of GABAergic neurons of the substantia nigra pars reticulata results in suppression of seizures in various animal models of epilepsy. The role in this control mechanism of the direct GABAergic projection from the striatum to the substantia nigra and of the indirect pathway, from the striatum through the globus pallidus and the subthalamic nucleus, was examined in a genetic model of absence seizures in the rat. In this model, pharmacological manipulations of both the direct and indirect pathways resulted in modulation of absence seizures. Activation of the direct pathway or inhibition of the indirect pathway suppressed absence seizures through disinhibition of neurons in the deep and intermediate layers of the superior colliculus. Dopamine D1 and D2 receptors in the nucleus accumbens, appear to be critical in these suppressive effects. Along with data from the literature, our results suggest that basal ganglia circuits play a major role in the modulation of absence seizures and provide a framework to understand the role of these circuits in the modulation of generalized seizures.

Role of the subthalamo-nigral input in the control of amygdala-kindled seizures in the rat.

The substantia nigra pars reticulata (SNpr) is a critical site for the control of epileptic seizures. Potentiation of the inhibitory GABAergic input from the striatum to the SNpr suppresses primary or secondary generalized seizures in the rat. The purpose of this study was to examine the possible involvement of the excitatory glutamatergic input from the subthalamic nucleus to the SNpr in the control of both the electroencephalographic and the motor components of amygdala-kindled seizures in the rat. Microinjections of either an N-methyl-D-aspartate (NMDA) antagonist in the substantia nigra or a GABAA agonist in the subthalamic nucleus, significantly reduced motor seizures but did not modified the afterdischarges. These results provide evidence for the involvement of the subthalamo-nigral projection in the modulation and the propagation of the motor components of amygdala-kindled seizures.

High-frequency stimulation of the subthalamic nucleus suppresses absence seizures in the rat: comparison with neurotoxic lesions.

High-frequency electrical stimulation of deep brain structures has recently been developed for the surgical approach of neurologic disorders. Applied to the thalamus in tremors or to the subthalamic nucleus in Parkinson's disease, high-frequency stimulation has been demonstrated to exert a local inhibiting influence, leading to symptoms alleviation. In the present study, bilateral high-frequency stimulations (130 Hz) of the subthalamic nuclei suppressed ongoing spontaneous absence seizures in rats. This effect was dissociated from motor side-effects and appears specific to the subthalamic nucleus. Bilateral excitotoxic lesions of the subthalamic nuclei only partially suppressed absence-seizures. These results confirm the involvement of the basal ganglia system in the control of generalized seizures and suggest that high-frequency stimulations could be used in the treatment of some forms of seizures.

Pathophysiological mechanisms of genetic absence epilepsy in the rat.

Generalized non-convulsive absence seizures are characterized by the occurrence of synchronous and bilateral spike and wave discharges (SWDs) on the electroencephalogram, that are concomitant with a behavioral arrest. Many similarities between rodent and human absence seizures support the use of genetic rodent models, in which spontaneous SWDs occur. This review summarizes data obtained on the neurophysiological and neurochemical mechanisms of absence seizures with special emphasis on the Genetic Absence Epilepsy Rats from Strasbourg (GAERS). EEG recordings from various brain regions and lesion experiments showed that the cortex, the reticular nucleus and the relay nuclei of the thalamus play a predominant role in the development of SWDs. Neither the cortex, nor the thalamus alone can sustain SWDs, indicating that both structures are intimely involved in the genesis of SWDs. Pharmacological data confirmed that both inhibitory and excitatory neurotransmissions are involved in the genesis and control of absence seizures. Whether the generation of SWDs is the result of an excessive cortical excitability, due to an unbalance between inhibition and excitation, or excessive thalamic oscillations, due to abnormal intrinsic neuronal properties under the control of inhibitory GABAergic mechanisms, remains controversial. The thalamo-cortical activity is regulated by several monoaminergic and cholinergic projections. An alteration of the activity of these different ascending inputs may induce a temporary inadequation of the functional state between the cortex and the thalamus and thus promote SWDs. The experimental data are discussed in view of these possible pathophysiological mechanisms.

[GABAergic mechanisms in generalized epilepsies: the neuroanatomical dimension]. / Mécanismes GABAergiques dans les épilepsies généralisées: la dimension neuroanatomique.

Generalized epileptic seizures are underlied by specific circuits where GABAergic synapses are involved at different levels. The role of these synapses depends on (i) the type of epilepsy and (ii) their localization within the central nervous system. This dual complexity can be illustrated by two examples from animal experimentation. Clinical, as well as experimental data have shown that the neural mechanisms underlying generalized non-convulsive seizures (e.g., absence-epilepsy) are distinct from those involved in convulsive generalized seizures. Pharmacological reactivity to anti-epileptic compounds is different between these two forms of seizures. Hippocampus and amygdala are key-structures in convulsive seizures whereas they are not involved in absence-epilepsy. A thalamo-cortical circuit generates the spike-and-wave discharges in absence epilepsy. Global activation of GABAergic transmission by systemic administration generally suppresses convulsive seizures whereas it aggravates absence in both humans and animals. Further investigations using a genetic model of absence seizures in the rat have suggested that this aggravation may be related to the role of post-synaptic GABA-B receptors in slow hyperpolarization, in the relay nuclei of the thalamus. By "de-inactivating" low-threshold calcium currents, activation of these receptors facilitates rhythmic activity in the thalamo-cortical circuit. In addition, regulation of transmitter release by presynaptic GABA-B receptors in the thalamus and the cortex may also contribute to the control of absence seizures. A blockade of the GABA-B receptors, either locally in the thalamus or systemically suppresses absence seizures. The critical role of the substantia nigra in the control of different forms of seizures has been demonstrated recently in the rat. This structure is one of the richest regions of the brain for GABAergic terminals, neurons and receptors. Local applications of GABA mimetics resulting in the desinhibition of their target neurons in the superior colliculus were shown to suppress both convulsive and non-convulsive seizures. This circuitry involving the basal ganglia may exert a "remote inhibitory control" over generalized epilepsies generated in other areas. In conclusion, the pharmacological manipulation of GABAergic transmission has different consequences on epilepsy depending on the form of seizures and the connections and functions of the GABAergic neurons in a given structure. The design of new therapeutical tools based on the manipulation of GABAergic mechanisms in the central nervous system requires to take into account this neuroanatomical dimension.

Anxiogenic-like consequences in animal models of complex partial seizures.

Several kinds of psychiatric symptoms (anxiety, depression, schizophrenia) have been associated with epilepsies, and clinical data suggest that patients with seizures involving limbic structures are the most prone to develop behavioural disorders between the seizures (i.e. interictally). Studying the neurobiological mechanisms that underlie these symptoms is difficult in humans because of different interfering factors (e.g. psychosocial difficulties, pharmacological side-effects, lesions), which can be avoided in animal models. Using repetitive electrical stimulations (kindling) or local applications of a neuroexcitotoxin in limbic structures (mainly the amygdala and hippocampus), several authors have reported lasting changes of emotional reactivity in cats and rats. These changes appear as anxiety-related reactions expressed as a hyperdefensiveness in the cat, or a reduction of spontaneous exploration in tests predictive of anxiogenic effects in the rat. Some neuroplasticity processes known to develop during epileptogenesis (neuronal-hyperexcitability, modulation of GABA/benzodiazepine transmission) may participate in these lasting changes of behaviour, especially in structures involved in the control of fear-promoted reactions (amygdala, periaqueductal grey matter). In addition, endogenous control systems may also play a critical role in the occurrence of interictal behavioural disorders.

Amygdala kindling in the rat: anxiogenic-like consequences.

Patients with complex partial seizures of temporal lobe origin may experience behavioural disorders like depressive, anxiety-related or schizophrenic-like symptoms between seizures, i.e. interictally. The neural mechanisms underlying these enduring interictal disorders remain to be investigated. The aim of the present study was to examine the behavioural consequences of kindling of the basolateral nuclei of the amygdala, an animal model of limbic complex partial seizures. Animals having experienced 15 stage 5 seizures were compared to non-kindled controls in different behavioural tests performed at least seven days after the last seizure. Kindled animals showed a significant reduction of exploration of open arms in the elevated plus-maze test. In the social interactions test, they showed a decrease of non-social behaviour and an increase of immobility. No modifications were observed in kindled animals when tested in the open field, the sucrose preference or the forced swimming test. The reduction of open arm exploration in the elevated plus-maze was reversed by a pretreatment with chlordiazepoxide (2 mg/kg i.p.), a benzodiazepine anxiolytic. Finally, a similar reduction of open arm exploration was observed when animals were kindled only until a stage 3 seizure occurred. These data, along with previous studies, suggest that kindling of the amygdala has anxiogenic consequences and provide an animal model to study the neuroplasticity phenomena underlying enduring interictal disorders in humans.

Involvement of nigral glutamatergic inputs in the control of seizures in a genetic model of absence epilepsy in the rat.

The reticular part of the substantia nigra is known to be a critical site in the control of epileptic seizures. Potentiation of the direct striatonigral GABAergic projection has been shown to suppress seizures in different animal models of epilepsy. Besides this GABAergic input, the substantia nigra receives glutamatergic inputs, especially from the indirect striatonigral pathway, via the subthalamic nucleus. To investigate the involvement of the nigral excitatory amino acid transmission in the remote control of non-convulsive generalized seizures, several drugs interacting with glutamatergic receptors were first injected into the substantia nigra pars reticulata in rats with spontaneous absence seizures. Blockade of N-methyl-D-aspartate receptors suppressed spontaneous generalized non-convulsive seizures in the rat, whereas blockade of non-N-methyl-D-aspartate receptors was without effect. Second, inhibition of the subthalamic projection by bilateral injections of a GABAergic agonist in this structure similarly suppressed absence seizures. These results suggest that excitatory amino acid inputs are critical in the triggering of the nigral control of generalized epilepsies. Furthermore, they support the hypothesis of a possible involvement of the subthalamonigral pathway in the control of generalized non-convulsive seizures.