Quentin J. Pittman

Epilepsy and brain inflammation

During the last decade, experimental research has demonstrated a prominent role of glial cells, activated in brain by various injuries, in the mechanisms of seizure precipitation and recurrence. In particular, alterations in the phenotype and function of activated astrocytes and microglial cells have been described in experimental and human epileptic tissue, including modifications in potassium and water channels, alterations of glutamine/glutamate cycle, changes in glutamate receptor expression and transporters, release of neuromodulatory molecules (e.g. gliotransmitters, neurotrophic factors), and induction of molecules involved in inflammatory processes (e.g. cytokines, chemokines, prostaglandins, complement factors, cell adhesion molecules) (Seifert et al., 2006; Vezzani et al., 2011; Wetherington et al., 2008). In particular, brain injury or proconvulsant events can activate microglia and astrocytes to release a number of proinflammatory mediators, thus initiating a cascade of inflammatory processes in brain tissue. Proinflammatory molecules can alter neuronal excitability and affect the physiological functions of glia by paracrine or autocrine actions, thus perturbing the glioneuronal communications. In experimental models, these changes contribute to decreasing the threshold to seizures and may compromise neuronal survival (Riazi et al., 2010; Vezzani et al., 2008). In this context, understanding which are the soluble mediators and the molecular mechanisms crucially involved in glio-neuronal interactions is instrumental to shed light on how brain inflammation may contribute to neuronal hyperexcitability in epilepsy. This review will report the clinical observations in drug-resistant human epilepsies and the experimental findings in adult and immature rodents linking brain inflammation to the epileptic process in a causal and reciprocal manner. By confronting the clinical evidence with the experimental findings, we will discuss the role of specific soluble inflammatory mediators in the etiopathogenesis of seizures, reporting evidence for both their acute and long term effects on seizure threshold. The possible contribution of these mediators to co-morbidities often described in epilepsy patients will be also discussed. Finally, we will report on the anti-inflammatory treatments with anticonvulsant actions in experimental models highlighting possible therapeutic options for treating drug-resistant seizures and for prevention of epileptogenesis.

Hypothalamic enkephalin neurones may regulate the neurohypophysis.

WE have previously reported that significant amounts of immunoreactive (ir)-Leu5-enkephalin were present in extracts of the neurointermediate lobe of the rat pituitary1. Negligible amounts of the pentapeptide were detected in the anterior lobe. In these assays, the concentration of Leu5-enkephalin in the neurointermediate lobe was higher than in the globus pallidus, the brain region reported to contain the densest enkephalinergic innervation2. The high content of (ir)-Leu-enkephalin in the neurointermediate lobe of the pituitary led us to further investigation of its distribution and possible function. We report here that (ir)-enkephalins in the pituitary are concentrated in nerve fibres projecting from the hypothalamus to the pars nervosa and that this pathway may be involved in the regulation of neurohypophysial neurosecretion.

Simultaneous Microdialysis in Blood and Brain: Oxytocin and Vasopressin Release in Response to Central and Peripheral Osmotic Stimulation and Suckling in the Rat

Simultaneous microdialysis in blood and brain has been used to monitor the release of both oxytocin and vasopressin into the systemic circulation (jugular vein/right atrium) and within the hypothalamic supraoptic nucleus of rats. Both home-made probes for blood and brain microdialysis revealed detectable nonapeptide concentrations under basal conditions and differential responses to a variety of stimuli. In urethane-anesthetized male rats, bilateral stimulation of the supraoptic nucleus by microdialyzing hypertonic medium (1 M NaCl) not only significantly increased the intranuclear release of both oxytocin and vasopressin (p < 0.05), but also their release from the neurohypophysis into blood (p < 0.05). In poststimulation microdialysates sampled from blood, the nonapeptides reached basal levels again, whereas intranuclear levels were further elevated. Intraperitoneal injection of hypertonic saline, on the other hand, resulted not only in the well-known increased peripheral release of oxytocin and vasopressin (p < 0.01 each), but also in a delayed increase in intranuclear oxytocin (p < 0.05). In contrast, intranuclear vasopressin release failed to change within the 90-min period following osmotic stimulation. In conscious lactating rats, suckling increased oxytocin contents in microdialysates sampled simultaneously in blood and the supraoptic nucleus (p < 0.05 each) further validating the microdialysis techniques used. The in vivo recovery in blood of approximately 65% determined using both radiolabeled and endogenous oxytocin provides a rough estimate to assess nonapeptide concentrations in plasma from 30-min or even 10-min blood microdialysis data.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats

There are critical postnatal periods during which even subtle interventions can have long-lasting effects on adult physiology. We asked whether an immune challenge during early postnatal development can alter neuronal excitability and seizure susceptibility in adults. Postnatal day 14 (P14) male Sprague Dawley rats were injected with the bacterial endotoxin lipopolysaccharide (LPS), and control animals received sterile saline. Three weeks later, extracellular recordings from hippocampal slices revealed enhanced field EPSP slopes after Schaffer collateral stimulation and increased epileptiform burst-firing activity in CA1 after 4-aminopyridine application. Six to 8 weeks after postnatal LPS injection, seizure susceptibility was assessed in response to lithium–pilocarpine, kainic acid, and pentylenetetrazol. Rats treated with LPS showed significantly greater adult seizure susceptibility to all convulsants, as well as increased cytokine release and enhanced neuronal degeneration within the hippocampus after limbic seizures. These persistent increases in seizure susceptibility occurred only when LPS was given during a critical postnatal period (P7 and P14) and not before (P1) or after (P20). This early effect of LPS on adult seizures was blocked by concurrent intracerebroventricular administration of a tumor necrosis factor α (TNFα) antibody and mimicked by intracerebroventricular injection of rat recombinant TNFα. Postnatal LPS injection did not result in permanent changes in microglial (Iba1) activity or hippocampal cytokine [IL-1β (interleukin-1β) and TNFα] levels, but caused a slight increase in astrocyte (GFAP) numbers. These novel results indicate that a single LPS injection during a critical postnatal period causes a long-lasting increase in seizure susceptibility that is strongly dependent on TNFα.

Microglial activation and TNFα production mediate altered CNS excitability following peripheral inflammation

Peripheral inflammation leads to a number of centrally mediated physiological and behavioral changes. The underlying mechanisms and the signaling pathways involved in these phenomena are not yet well understood. We hypothesized that peripheral inflammation leads to increased neuronal excitability arising from a CNS immune response. We induced inflammation in the gut by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to adult male rats. To examine the excitability of the brain in vivo, we administered pentylenetetrazole (PTZ; a GABAergic antagonist) intravenously to evoke clonic seizures. Rats treated with TNBS showed increased susceptibility to PTZ seizures that was strongly correlated with the severity and progression of intestinal inflammation. In vitro hippocampal slices from inflamed, TNBS-treated rats showed increased spontaneous interictal burst firing following application of 4-aminopyridine, indicating increased intrinsic excitability. The TNBS-treated rats exhibited a marked, reversible inflammatory response within the hippocampus, characterized by microglial activation and increases in tumor necrosis factor α (TNFα) levels. Central antagonism of TNFα using a monoclonal antibody or inhibition of microglial activation by i.c.v. injection of minocycline prevented the increase in seizure susceptibility. Moreover, i.c.v. infusion of TNFα in untreated rats for 4 days also increased seizure susceptibility and thus mimicked the changes in seizure threshold observed with intestinal inflammation. Our finding of a microglia-dependent TNFα-mediated increase in CNS excitability provides insight into potential mechanisms underlying the disparate neurological and behavioral changes associated with chronic inflammation.

Talking back: dendritic neurotransmitter release

Classical transmitters and neuropeptides can be released from the dendrites of many neuronal populations, to act as retrograde signals that modulate synaptic transmission, electrical activity and, in some cases, morphology of the cell of origin. For the hypothalamic neuroendocrine cells that release vasopressin and oxytocin, the stimuli, mechanisms and physiological functions of dendritic release have been revealed in detail that is not yet available for other neurons. The regulation of dendritic transmitter release is complex and at least partially independent from axon terminal release. Here, we provide an overview of recent findings on the mechanisms and physiological consequences of dendritic neuropeptide release and place this in the context of discoveries of dendritic neurotransmitter release in other brain regions.

Somatostatin hyperpolarizes hippocampal pyramidal cells in vitro

Superfusion onto hippocampal slices of low concentrations of the tetradecapeptide somatostatin (SS), or an SS analogue having CNS activity, reversibly hyperpolarized pyramidal neurons, as revealed by intracellular recording. The hyperpolarizations were accompanied by reductions in spontaneous and evoked spike discharge and in input resistance; the magnitude of the hyperpolarizations was not influenced by 10 mM MgCl2 added to the perfusate to block synaptic transmission.

Dendritically Released Peptides Act as Retrograde Modulators of Afferent Excitation in the Supraoptic Nucleus In Vitro

Oxytocin (OXT) and vasopressin (VP) are known to be released from dendrites of magnocellular neurons. Here, we show that these peptides reduced evoked EPSCs by a presynaptic mechanism, an effect blocked by peptide antagonists and mimicked by inhibition of endogenous peptidases. Dendritic release of peptides, elicited with depolarization achieved by high frequency stimulation of afferents or with current injection into an individual neuron, induced short-term synaptic depression similar to that seen following exogenous peptide application and was prevented by peptide antagonists. Thus, dendritically released peptides depress evoked EPSCs in magnocellular neurons by activating presynaptic OXT and/or VP receptors. Such a retrograde modulatory action on afferent excitation may serve as a feedback mechanism to permit peptidergic neurosecretory neurons to autoregulate their own activity.

Contributions of peripheral inflammation to seizure susceptibility: Cytokines and brain excitability

Inflammation is an important factor in the pathophysiology of seizure generation and epileptogenesis. While the role of CNS inflammation is well acknowledged as an important factor in seizure pathophysiology, less is known about the role of peripheral inflammation. Systemic inflammation induces a mirror inflammatory response in the brain that might have transient or long-term effects on seizure susceptibility. The focus of our laboratory research is the study of the interaction of systemic inflammatory events with neuronal excitability and seizure susceptibility. In this paper we provide a review of our findings and discuss possible mechanisms.

Presynaptic action of neuropeptide Y in area CA1 of the rat hippocampal slice.

1. Neuropeptide tyrosine (neuropeptide Y, NPY), a recently isolated endogenous brain peptide, reduces the extracellular population spike evoked by stimulation of stratum radiatum in area CA1 of the in vitro rat hippocampal slice, without reducing the antidromically evoked population spike. To test the hypothesis that NPY acts presynaptically, intracellular recordings were made of pyramidal neurones of area CA1 in vitro. 2. Bath application of 10(‐6) M‐NPY causes a long‐lasting (1‐1.5 h), reversible reduction of the orthodromically evoked excitatory post‐synaptic potential (e.p.s.p.) recorded intracellularly from CA1 pyramidal neurones. This effect on the e.p.s.p. was dependent upon the concentration of NPY. 3. The resting membrane potential, slope input resistance, and action potential threshold, amplitude and duration of the CA1 pyramidal neurones were not affected by NPY. 4. The responses of CA1 pyramidal neurones to ionophoretic pulses of glutamate, applied to the dendrites during synaptic blockade, was also unaffected by NPY. 5. The evidence supports the hypothesis that NPY acts presynaptically in the CA1 region of hippocampus to reduce excitatory input to the pyramidal neurones.