Ketogenic diet for mood disorders from animal models to clinical application.

Mood disorders such as major depressive disorder (MDD) and bipolar disorder (BD) are often resistant to current pharmacological treatment. Therefore, various alternative therapeutic approaches including diets are, therefore, under investigation. Ketogenic diet (KD) is effective for treatment-resistant epilepsy and metabolic diseases, however, only a few clinical studies suggest its beneficial effect also for mental disorders. Animal models are a useful tool to uncover the underlying mechanisms of therapeutic effects. Women have a twice-higher prevalence of mood disorders but very little is known about sex differences in nutritional psychiatry. In this review, we aim to summarize current knowledge of the sex-specific effects of KD in mood disorders. Ketone bodies improve mitochondrial functions and suppress oxidative stress, inducing neuroprotective and anti-inflammatory effects which are both beneficial for mental health. Limited data also suggest KD-induced improvement of monoaminergic circuits and hypothalamus-pituitary-adrenal axis-the key pathophysiological pathways of mood disorders. Gut microbiome is an important mediator of the beneficial and detrimental effects of diet on brain functioning and mental health. Gut microbiota composition is affected in mood disorders but its role in the therapeutic effects of different diets, including KD, remains poorly understood. Still little is known about sex differences in the effects of KD on mental health as well as on metabolism and body weight. Some animal studies used both sexes but did not find differences in behavior, body weight loss or gut microbiota composition. More studies, both on a preclinical and clinical level, are needed to better understand sex-specific effects of KD on mental health.

Refinement of the Barnes and Morris water maze protocols improves characterization of spatial cognitive deficits in the lithium-pilocarpine rat model of epilepsy.

Temporal lobe epilepsy (TLE) often causes cognitive impairment, especially a decline in spatial memory. Reductions in spatial memory and learning are also common in rodent models of TLE. The Morris water maze and the Barnes maze are the standard methods for evaluating spatial learning and memory in rodents. However, animals with TLE may exhibit agitation, distress, and fail to follow the paradigmatic context of these tests, making the interpretation of experimental data difficult. This study optimized the procedure of the Morris water maze and the Barnes maze to evaluate spatial learning and memory in rats with the lithium-pilocarpine TLE model (LPM rats). It was demonstrated that LPM rats required a mandatory and prolonged habituation stage for both tests. Therefore, the experimental rats performed relatively well on these tests. Nevertheless, LPM rats exhibited a slower learning process compared to the control rats. LPM rats also showed a reduction in spatial memory formation. This was more pronounced in the Barnes maze. Also, LPM rats utilized a sequential strategy for searching in the Barnes maze and were incapable of developing a more efficient spatial search strategy that is common in control animals. The Barnes maze may be a better choice for assessing search strategies, learning deficits, and spatial memory in rats with TLE when choosing between the two tests. This is because of the risk of unexpected seizure occurrence during the Morris water maze tests, and the potential risks for animal welfare.

MTEP, a Selective mGluR5 Antagonist, Had a Neuroprotective Effect but Did Not Prevent the Development of Spontaneous Recurrent Seizures and Behavioral Comorbidities in the Rat Lithium-Pilocarpine Model of Epilepsy.

Metabotropic glutamate receptors (mGluRs) are expressed predominantly on neurons and glial cells and are involved in the modulation of a wide range of signal transduction cascades. Therefore, different subtypes of mGluRs are considered a promising target for the treatment of various brain diseases. Previous studies have demonstrated the seizure-induced upregulation of mGluR5; however, its functional significance is still unclear. In the present study, we aimed to clarify the effect of treatment with the selective mGluR5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) on epileptogenesis and behavioral impairments in rats using the lithium-pilocarpine model. We found that the administration of MTEP during the latent phase of the model did not improve survival, prevent the development of epilepsy, or attenuate its manifestations in rats. However, MTEP treatment completely prevented neuronal loss and partially attenuated astrogliosis in the hippocampus. An increase in excitatory amino acid transporter 2 expression, which has been detected in treated rats, may prevent excitotoxicity and be a potential mechanism of neuroprotection. We also found that MTEP administration did not prevent the behavioral comorbidities such as depressive-like behavior, motor hyperactivity, reduction of exploratory behavior, and cognitive impairments typical in the lithium-pilocarpine model. Thus, despite the distinct neuroprotective effect, the MTEP treatment was ineffective in preventing epilepsy.

Ceftriaxone Treatment Weakens Long-Term Synaptic Potentiation in the Hippocampus of Young Rats.

Disrupted glutamate clearance in the synaptic cleft leads to synaptic dysfunction and neurological diseases. Decreased glutamate removal from the synaptic cleft is known to cause excitotoxicity. Data on the physiological effects of increased glutamate clearance are contradictory. This study investigated the consequences of ceftriaxone (CTX), an enhancer of glutamate transporter 1 expression, treatment on long-term synaptic potentiation (LTP) in the hippocampus of young rats. In this study, 5-day administration of CTX (200 mg/kg) significantly weakened LTP in CA3-CA1 synapses. As shown by electrophysiological recordings, LTP attenuation was associated with weakening of N-Methyl-D-aspartate receptor (NMDAR)-dependent signaling in synapses. However, PCR analysis did not show downregulation of NMDAR subunits or changes in the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits. We assume that extracellular burst stimulation activates fewer synapses in CTX-treated animals because increased glutamate reuptake results in reduced spillover, and neighboring synapses do not participate in neurotransmission. Attenuation of LTP was not accompanied by noticeable behavioral changes in the CTX group, with no behavioral abnormalities observed in the open field test or Morris water maze test. Thus, our experiments show that increased glutamate clearance can impair long-term synaptic plasticity and that this phenomenon can be considered a potential side effect of CTX treatment.

Exposure to bacterial lipopolysaccharidein early life affects the expression of ionotropic glutamate receptor genes and is accompanied by disturbances in long-term potentiation and cognitive functions in young rats.

Infections in childhood play an essential role in the pathogenesis of cognitive and psycho-emotional disorders. One of the possible mechanisms of these impairments is changes in the functional properties of NMDA and AMPA glutamate receptors in the brain. We suggest that bacterial infections during the early life period, which is critical for excitatory synapse maturation, can affect the subunit composition of NMDA and AMPA receptors. In the present study, we investigated the effect of repetitive lipopolysaccharide (LPS) intraperitoneal (i.p.) administration (25 µg/kg/day on P14, 16, and 18), mimicking an infectious disease, on the expression of subunits of NMDA and AMPA receptors in young rats. We revealed a substantial decrease of GluN2B subunit expression in the hippocampus at P23 using Western blot analysis and real-time polymerase chain reaction assay. Moderate changes were also found in GluN1, GluN2A, and GluA1 mRNA expression. The LPS-treated rats exhibited decreased exploratory and locomotor activity in the open field test and the impairment of spatial learning in the Morris water maze. Behavioral impairments were accompanied by a significant reduction in long-term hippocampal synaptic potentiation. Our data indicate that LPS-treatment in the critical period for excitatory synapse maturation alters ionotropic glutamate receptor gene expression, disturbs synaptic plasticity, and alters behavior.

Ceftriaxone Treatment Affects EAAT2 Expression and Glutamatergic Neurotransmission and Exerts a Weak Anticonvulsant Effect in Young Rats.

Epilepsy is a common neurological disorder. Despite the availability of a wide range of antiepileptic drugs, these are unsuccessful in preventing seizures in 20-30% of patients. Therefore, new pharmacological strategies are urgently required to control seizures. Modulation of glutamate uptake may have potential in the treatment of pharmacoresistant forms of epilepsy. Previous research showed that the antibiotic ceftriaxone (CTX) increased the expression and functional activity of excitatory amino acid transporter 2 (EAAT2) and exerted considerable anticonvulsant effects. However, other studies did not confirm a significant anticonvulsant effect of CTX administration. We investigated the impacts of CTX treatment on EAAT expression and glutamatergic neurotransmission, as well its anticonvulsant action, in young male Wistar rats. As shown by a quantitative real-time polymerase chain reaction (qPCR) assay and a Western blot analysis, the mRNA but not the protein level of EAAT2 increased in the hippocampus following CTX treatment. Repetitive CTX administration had only a mild anticonvulsant effect on pentylenetetrazol (PTZ)-induced convulsions in a maximal electroshock threshold test (MEST). CTX treatment did not affect the glutamatergic neurotransmission, including synaptic efficacy, short-term facilitation, or the summation of excitatory postsynaptic potentials (EPSPs) in the hippocampus and temporal cortex. However, it decreased the field EPSP (fEPSP) amplitudes evoked by intense electrical stimulation. In conclusion, in young rats, CTX treatment did not induce overexpression of EAAT2, therefore exerting only a weak antiseizure effect. Our data provide new insight into the effects of modulation of EAAT2 expression on brain functioning.

Neurobiology, Functions, and Relevance of Excitatory Amino Acid Transporters (EAATs) to Treatment of Refractory Epilepsy.

Epilepsy is one of the most prevalent and devastating neurological disorders characterized by episodes of unusual sensations, loss of awareness, and reoccurring seizures. The frequency and intensity of epileptic fits can vary to a great degree, with almost a third of all cases resistant to available therapies. At present, there is a major unmet need for effective and specific therapeutic intervention. Impairments of the exquisite balance between excitatory and inhibitory synaptic processes in the brain are considered key in the onset and pathophysiology of the disease. As the primary excitatory neurotransmitter in the central nervous system, glutamate has been implicated in the process, with the glutamatergic system holding center stage in the pathobiology as well as in developing disease-modifying therapies. Emerging data pinpoint impairments of glutamate clearance as one of the key causative factors in drug-resistant disease forms. Reinstatement of glutamate homeostasis using pharmacological and genetic modulation of glutamate clearance is therefore considered to be of major translational relevance. In this article, we review the neurobiological and clinical evidence suggesting complex aberrations in the activity and functions of excitatory amino acid transporters (EAATs) in epilepsy, with knock-on effects on glutamate homeostasis as a leading cause for the development of refractory forms. We consider the emerging data on pharmacological and genetic manipulations of EAATs, with reference to seizures and glutamate dyshomeostasis, and review their fundamental and translational relevance. We discuss the most recent advances in the EAATs research in human and animal models, along with numerous questions that remain open for debate and critical appraisal. Contrary to the widely held view on EAATs as a promising therapeutic target for management of refractory epilepsy as well as other neurological and psychiatric conditions related to glutamatergic hyperactivity and glutamate-induced cytotoxicity, we stress that the true relevance of EAAT2 as a target for medical intervention remains to be fully appreciated and verified. Despite decades of research, the emerging properties and functional characteristics of glutamate transporters and their relationship with neurophysiological and behavioral correlates of epilepsy challenge the current perception of this disease and fit unambiguously in neither EAATs functional deficit nor in reversal models. We stress the pressing need for new approaches and models for research and restoration of the physiological activity of glutamate transporters and synaptic transmission to achieve much needed therapeutic effects. The complex mechanism of EAATs regulation by multiple factors, including changes in the electrochemical environment and ionic gradients related to epileptic hyperactivity, impose major therapeutic challenges. As a final note, we consider the evolving views and present a cautious perspective on the key areas of future progress in the field towards better management and treatment of refractory disease forms.

Paradoxical Anticonvulsant Effect of Cefepime in the Pentylenetetrazole Model of Seizures in Rats.

Many ß-lactam antibiotics, including cephalosporins, may cause neurotoxic and proconvulsant effects. The main molecular mechanism of such effects is considered to be γ-aminobutyric acid type a (GABAa) receptor blockade, leading to the suppression of GABAergic inhibition and subsequent overexcitation. We found that cefepime (CFP), a cephalosporin, has a pronounced antiepileptic effect in the pentylenetetrazole (PTZ)-induced seizure model by decreasing the duration and severity of the seizure and animal mortality. This effect was specific to the PTZ model. In line with findings of previous studies, CFP exhibited a proconvulsant effect in other models, including the maximal electroshock model and 4-aminopyridine model of epileptiform activity, in vitro. To determine the antiepileptic mechanism of CFP in the PTZ model, we used whole-cell patch-clamp recordings. We demonstrated that CFP or PTZ decreased the amplitude of GABAa receptor-mediated postsynaptic currents. PTZ also decreased the current decay time constant and temporal summation of synaptic responses. In contrast, CFP slightly increased the decay time constant and did not affect summation. When applied together, CFP prevented alterations to the summation of responses by PTZ, strongly reducing the effects of PTZ on repetitive inhibitory synaptic transmission. The latter may explain the antiepileptic effect of CFP in the PTZ model.

Anakinra Reduces Epileptogenesis, Provides Neuroprotection, and Attenuates Behavioral Impairments in Rats in the Lithium-Pilocarpine Model of Epilepsy.

Temporal lobe epilepsy is a widespread chronic disorder that manifests as spontaneous seizures and is often characterized by refractoriness to drug treatment. Temporal lobe epilepsy can be caused by a primary brain injury; therefore, the prevention of epileptogenesis after a primary event is considered one of the best treatment options. However, a preventive treatment for epilepsy still does not exist. Neuroinflammation is directly involved in epileptogenesis and neurodegeneration, leading to the epileptic condition and cognitive decline. In the present study, we aimed to clarify the effect of treatment with a recombinant form of the Interleukin-1 receptor antagonist (anakinra) on epileptogenesis and behavioral impairments in rats using the lithium-pilocarpine model. We found that anakinra administration during the latent phase of the model significantly suppressed the duration and frequency of spontaneous recurrent seizures in the chronic phase. Moreover, anakinra administration prevented some behavioral impairments, including motor hyperactivity and disturbances in social interactions, during both the latent and chronic periods. Histological analysis revealed that anakinra administration decreased neuronal loss in the CA1 and CA3 areas of the hippocampus but did not prevent astro- and microgliosis. The treatment increased the expression level of the solute carrier family 1 member 2 gene (Slc1a2, encoding excitatory amino acid transporter 2 (EAAT2)) in the hippocampus, potentially leading to a neuroprotective effect. However, the increased gene expression of proinflammatory cytokine genes (Interleukin-1ß (Il1b) and tumor necrosis factor α (Tnfa)) and astroglial marker genes (glial fibrillary acidic protein (Gfap) and inositol 1,4,5-trisphosphate receptor type 2 (Itpr2)) in experimental rats was not affected by anakinra treatment. Thus, our data demonstrate that the administration of anakinra during epileptogenesis has some beneficial disease-modifying effects.

Changes in Functional Properties of Rat Hippocampal Neurons Following Pentylenetetrazole-induced Status Epilepticus.

Pathophysiological remodeling processes following status epilepticus (SE) play a critical role in the pathophysiology of epilepsy but have not yet been not fully investigated. In the present study, we examined changes in intrinsic properties of pyramidal neurons, basal excitatory synaptic transmission, and short-term synaptic plasticity in hippocampal slices of rats after SE. Seizures were induced in 3-week-old rats by an intraperitoneal pentylenetetrazole (PTZ) injection. Only animals with generalized seizures lasting more than 30 min were included in the experiments. We found that CA1 pyramidal neurons became more excitable and started firing at a lower excitatory input due to a significant increase in input resistance. However, basal excitatory synaptic transmission was reduced in CA3-CA1 synapses, thus preventing the propagation of excitation through neural networks. A significant increase in paired-pulse facilitation 1 d after SE pointed to a decrease in the probability of glutamate release. Increased intrinsic excitability of neurons and decreased synaptic transmission differentially affected the excitability of a neural network. In terms of changes in seizure susceptibility after SE, we observed a significant increase in the maximal electroshock threshold 1 day after SE, suggesting a decrease in seizure susceptibility. However, after 1 week, there was no difference in seizure susceptibility between control and post-SE rats. The effects of SE on functional properties of hippocampal neurons were transient in the PTZ model, and most of them had recovered 1 week after SE. However, some minor alterations, such as smaller amplitude field potentials, were observed 1 month after SE.

Impairments in cognitive functions and emotional and social behaviors in a rat lithium-pilocarpine model of temporal lobe epilepsy.

The aim of this study was to evaluate in detail behavioral patterns and comorbid disturbances in rats using the lithium-pilocarpine model. A comprehensive set of behavioral tests was used to investigate behavioral patterns, including the open field test, Morris water maze, Y-maze, fear conditioning, the elevated plus maze, the forced swimming test, and the resident-intruder paradigm. Motor and explorative activity, learning and memory, anxiety and depressive-like behavior, aggression, and communication were evaluated 8-15 d after pilocarpine-induced status epilepticus (SE) (latent phase of the model) and 41-53 d (chronic phase) after pilocarpine-induced SE. Increased motor activity and impaired memory function were the most noticeable behavioral modifications in the epileptic rats. Both the movement speed and distance traveled increased in the open field test in both the latent and chronic phases. Significant impairments were detected in short-and long-term spatial memory in the Morris water maze during the latent phase. Besides the alterations in spatial memory, behaviors indicative of short- and long-term fear-associated memory disturbances were observed in the fear conditioning test during the chronic phase of the model. In the resident-intruder paradigm, epileptic rats exhibited disturbed communicative behavior, with impaired social behaviors. In contrast, emotional disturbances were less prominent, with the rats exhibiting decreased anxiety. There were no changes in depressive-like behavior. The data suggest that the lithium-pilocarpine model of TLE in rodents is more useful for studies of comorbid disturbances in memory, hyperactivity, and social behavior than for research on psychoemotional impairments, such as anxiety and depression.