Roland S. G. Jones

The pilocarpine model of temporal lobe epilepsy

Understanding the pathophysiogenesis of temporal lobe epilepsy (TLE) largely rests on the use of models of status epilepticus (SE), as in the case of the pilocarpine model. The main features of TLE are: (i) epileptic foci in the limbic system; (ii) an “initial precipitating injury”; (iii) the so-called “latent period”; and (iv) the presence of hippocampal sclerosis leading to reorganization of neuronal networks. Many of these characteristics can be reproduced in rodents by systemic injection of pilocarpine; in this animal model, SE is followed by a latent period and later by the appearance of spontaneous recurrent seizures (SRSs). These processes are, however, influenced by experimental conditions such as rodent species, strain, gender, age, doses and routes of pilocarpine administration, as well as combinations with other drugs administered before and/or after SE. In the attempt to limit these sources of variability, we evaluated the methodological procedures used by several investigators in the pilocarpine model; in particular, we have focused on the behavioural, electrophysiological and histopathological findings obtained with different protocols. We addressed the various experimental approaches published to date, by comparing mortality rates, onset of SRSs, neuronal damage, and network reorganization. Based on the evidence reviewed here, we propose that the pilocarpine model can be a valuable tool to investigate the mechanisms involved in TLE, and even more so when standardized to reduce mortality at the time of pilocarpine injection, differences in latent period duration, variability in the lesion extent, and SRS frequency.

Entorhinal-hippocampal connections: a speculative view of their function

On the basis of neuroanatomical studies, the entorhinal cortex (EC) has long been regarded as a relay station that provides the major source of afferent input to the hippocampus. The perforant path input to the dentate gyrus from layer II has traditionally been regarded as the major pathway by which information is transferred. However, electrophysiological studies are now indicating that other elements of the perforant path that project directly to CA1 and CA3 are more important than thought previously, and that the properties of different neuronal elements in the EC may determine the way in which information is passed on to and processed by the hippocampus. This article summarizes some of the properties of synaptic transmission in the EC and speculates on how frequency-dependent changes in transmission may be involved in the pre- and post-processing of hippocampal information by the EC.

The anticonvulsant, lamotrigine decreases spontaneous glutamate release but increases spontaneous GABA release in the rat entorhinal cortex in vitro.

It has been suggested that the anticonvulsant effect of lamotrigine resides with its ability to block voltage gated Na-channels at presynaptic sites, thus stabilizing the presynapse, and, consequently, reducing the release of synaptic transmitters. Neurochemical studies have shown that it can inhibit the veratrine-stimulated release of the excitatory transmitter, glutamate from cortical tissue, but that at slightly higher concentrations it also reduces the release of the inhibitory transmitter, GABA. In the present study we examined the effect of the drug on the release of these transmitters at synapses in the rat entorhinal cortex, using the whole-cell patch clamp technique to record spontaneous excitatory (EPSCs) and inhibitory postsynaptic currents (IPSCs). Lamotrigine reduced the frequency, but not the amplitude of spontaneous EPSCs. This clearly indicated a presynaptic effect to reduce the release of glutamate. However, the same effect was observed when we tested the drug on miniature EPSCs, recorded in the presence of TTX and Cd, showing that blockade of Na-channels or Ca-channels was not a prerequisite for inhibition of glutamate release. In contrast to its effects on EPSCs, lamotrigine increased both the frequency and amplitude of spontaneous IPSCs, suggesting that the drug was acting presynaptically to enhance GABA release. Again, similar effects were seen with miniature IPSCs recorded in TTX. These opposite effects of lamotrigine on glutamate and GABA release are similar to those we have reported previously with phenytoin, and suggest that reciprocal modulation of the background release of the major excitatory and inhibitory transmitters may be a significant factor in dampening excitability in pathologically hyperexcitable cortical networks.

Laminar differences in recurrent excitatory transmission in the rat entorhinal cortex in vitro

Paired intracellular recordings were used to investigate recurrent excitatory transmission in layers II, III and V of the rat entorhinal cortex in vitro. There was a relatively high probability of finding a recurrent connection between pairs of pyramidal neurons in both layer V (around 12%) and layer III (around 9%). In complete contrast, we have failed to find any recurrent synaptic connections between principal neurons in layer II, and this may be an important factor in the relative resistance of this layer in generating synchronized epileptiform activity. In general, recurrent excitatory postsynaptic potentials in layers III and V of the entorhinal cortex had similar properties to those recorded in other cortical areas, although the probabilities of connection are among the highest reported. Recurrent excitatory postsynaptic potentials recorded in layer V were smaller with faster rise times than those recorded in layer III. In both layers, the recurrent potentials were mediated by glutamate primarily acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazole receptors, although there appeared to be a slow component mediated by N-methyl-D-aspartate receptors. In layer III, recurrent transmission failed on about 30% of presynaptic action potentials evoked at 0.2Hz. This failure rate increased markedly with increasing (2, 3Hz) frequency of activation. In layer V the failure rate at low frequency was less (19%), and although it increased at higher frequencies this effect was less pronounced than in layer III. Finally, in layer III, there was evidence for a relatively high probability of electrical coupling between pyramidal neurons. We have previously suggested that layers IV/V of the entorhinal cortex readily generate synchronized epileptiform discharges, whereas layer II is relatively resistant to seizure generation. The present demonstration that recurrent excitatory connections are widespread in layer V but not layer II could support this proposal. The relatively high degree of recurrent connections and electrical coupling between layer III cells may be a factor in its susceptibility to neurodegeneration during chronic epileptic conditions.

Dual effects of gabapentin and pregabalin on glutamate release at rat entorhinal synapses in vitro.

We have recently shown that the anticonvulsant drugs phenytoin, lamotrigine and sodium valproate all reduce the release of glutamate at synapses in the entorhinal cortex in vitro. In the present investigation we determined whether this property was shared by gabapentin and pregabalin, using whole‐cell patch‐clamp recordings of excitatory postsynaptic currents (EPSCs) in layer V neurons in slices of rat entorhinal cortex. Both drugs reduced the amplitude and increased the paired‐pulse ratio of EPSCs evoked by electrical stimulation of afferent inputs, suggesting a presynaptic effect to reduce glutamate release. The frequency of spontaneous EPSCs (sEPSCs) was concurrently reduced by GBP, further supporting a presynaptic action. There was no significant change in amplitude although a slight reduction was seen, particularly with gabapentin, which may reflect a reduction in the number of larger amplitude sEPSCs. When activity‐independent miniature EPSCs were recorded in the presence of tetrodotoxin, both drugs continued to reduce the frequency of events with no change in amplitude. The reduction in frequency induced by gabapentin or pregabalin was blocked by application of the l‐amino acid transporter substrate l‐isoleucine. The results show that gabapentin and pregabalin, like other anticonvulsants, reduce glutamate release at cortical synapses. It is possible that this reduction is a combination of two effects: a reduction of activity‐dependent release possibly via interaction with P/Q‐type voltage‐gated Ca channels, and a second action, as yet unidentified, occurring downstream of Ca influx into the presynaptic terminals.

Synchronous discharges in the rat entorhinal cortex in vitro: Site of initiation and the role of excitatory amino acid receptors

A slice preparation was used to study the spread of epileptiform activity in the rat entorhinal cortex. Interictal-like discharges were induced in the medial entorhinal cortex by blocking synaptic inhibition mediated via GABAA-receptors. Recorded intracellularly, these discharges consisted of an initial paroxysmal depolarizing shift followed by a variable number of afterdischarges. There was no apparent difference between these events whether they were recorded in isolated cortical slices or in slices where the hippocampus and subicular complex remained attached. The events were also unaffected by droplets of a xylocaine solution applied to sites in the hippocampus, subicular complex or superficial layers of the entorhinal cortex but applications to layer IV/V, lateral or medial to the recording site could reduce the number of afterdischarges without affecting the initial paroxysmal shift. Simultaneous intracellular recordings from neurons in layer IV/V and layer II of the medial entorhinal cortex showed that the paroxysmal depolarizing shift and all afterdischarges in the deeper layer always preceded those recorded in the superficial layer, and these events invariably occurred on a one-to-one basis. This was true whether the events were evoked or occurred spontaneously. The delay varied between 2 and 11 ms but was consistent for a given cell pair. A similar relationship existed between discharges recorded simultaneously in layer IV/V neurons and layer VI neurons, events in the layer IV/V cells preceding those in the deeper layer. Discharges recorded simultaneously in pairs of layer IV/V neurons showed more complex relationships. Paroxysmal depolarizing shifts were always recorded in both cells and the discharge could occur at the more medial site before the more lateral, or vice versa. For a given pair the temporal relationship was invariable. It was often the case, however, that the temporal relationship between afterdischarges was reversed with respect to the initial paroxysmal shift. This relationship was also invariable in a given pair of cells. Interictal-like discharges in layers II or IV/V neurons could be abolished by perfusion with 6-cyano-7-nitro-quinoxaline-2,3-dione which is an antagonist for the non-N-methyl-D-aspartate (i.e. quisqualate/kainate) subtype of excitatory amino acid receptor. The afterdischarges associated with the events were abolished in an all-or-none fashion whereas the blockade of the paroxysmal depolarizing shift was progressive. Antagonists of N-methyl-D-aspartate receptors also abolished afterdischarges but only reduced the initial paroxysmal shift. It is concluded that the interictal-like discharges arise intrinsically within the cortex and are not influenced by input from hippocampal or subicular structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Tonic Facilitation of Glutamate Release by Presynaptic NR2B-Containing NMDA Receptors Is Increased in the Entorhinal Cortex of Chronically Epileptic Rats

We have shown previously that when postsynaptic NMDA receptors are blocked, the frequency, but not amplitude, of spontaneous EPSCs (sEPSCs) at synapses in the entorhinal cortex is reduced by NMDA receptor antagonists, demonstrating that glutamate release is tonically facilitated by presynaptic NMDA autoreceptors. In the present study, we recorded sEPSCs using whole-cell voltage clamp in neurons in layer V in slices of the rat entorhinal cortex. Using specific antagonists for NR2A [(R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid] and NR2B [(αR, βS)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol hydrochloride (Ro 25-6981)] subunit-containing receptors, we confirmed that in slices from juvenile rats (4–6 weeks of age), the autoreceptor is predominantly of the NR1–NR2B subtype. In older (4–6 months of age) control animals, the effect of the NR2B antagonist was less marked, suggesting a decline in autoreceptor function with development. In slices from rats (aged 4–6 months) exhibiting spontaneous recurrent seizures induced with a lithium-pilocarpine protocol, Ro 25-6981 again robustly reduced sEPSC frequency. The effect was equal to or greater than that seen in the juvenile slices and much more pronounced than that seen in the age-matched control animals. In all three groups, the NR2A antagonist was without effect on sEPSCs. These results suggest that there is a developmental decrease in NMDA autoreceptor function, which is reversed in a chronic epileptic condition. The enhanced autoreceptor function may contribute to seizure susceptibility and epileptogenesis in temporal lobe structures.

Interactions of Dopamine with Glutamate- and GABA-mediated Synaptic Transmission in the Rat Entorhinal Cortex In Vitro

We have studied the interactions between dopamine and glutamate‐mediated transmission in the entorhinal cortex using intracellular recording in a slice preparation from the rat brain. High concentrations (0.1 – 1 mM) of dopamine had weak, direct effects on the membrane potential with predominantly hyperpolarizing responses in layer II neurons and depolarizing responses in layer V. Studies with the dopamine antagonists sulpiride (D2 antagonist, 10 – 50 μM) and SCH‐23390 (D1 antagonist, 50 μM) indicated that the hyperpolarization by dopamine could be mediated by D2 receptors, although the pharmacology was not clear‐cut. The depolarizing response was not affected by either D1 or D2 antagonists. Synaptic responses of layer II and layer V cells were complex, consisting of both inhibitory and excitatory potentials. In untreated slices, dopamine reduced all components of the synaptic responses. However, when components of the responses were pharmacologically isolated, only the excitatory, glutamate‐mediated potentials were consistently affected and the GABAergic inhibitory potentials were more resistant to reduction by dopamine. Excitatory potentials mediated by both N‐methyl‐d‐aspartate and α‐amino‐3‐hydroxy‐5‐methyl‐isoxazolepropionic acid receptors were reduced by dopamine, but the former were more strongly affected. Studies with antagonists suggested that the D1 receptor is more likely to be involved in the decrement of glutamate transmission. Thus, dopamine appears to modulate glutamate‐mediated synaptic transmission in the entorhinal cortex, and it is conceivable that a disturbance in this interaction could be involved in the aetiology of schizophrenia.

Epileptiform events induced by GABA-antagonists in entorhinal cortical cells in vitro are partly mediated byN-methyl-d-aspartate receptors

The effects of the GABA-antagonists, picrotoxin and bicuculline on responses of medial entorhinal cortical cells to subicular stimulation were tested in vitro. On every cell tested either antagonist caused a profound enhancement of synaptically evoked depolarizations to the point where paroxysmal depolarizing shifts (PDS) were recorded, although only a minority of cells showed evidence of inhibitory potentials in the control situation. The initial PDS was followed by either a long afterdepolarization or a series of afterdischarges. The afterpotentials were always reduced or blocked by the N-methyl-D-aspartate (NMDA)-receptor antagonist, 2-amino-5-phosphonovalerate (2-AP5). The amplitude of the initial PDS in many cells was also reduced by 2-AP5. Thus, entorhinal cortical cells are susceptible to epileptogenesis induced by a reduction of GABAergic inhibition and the paroxysmal events contain a large, NMDA-receptor mediated component.

Electrophysiological characterisation of tachykinin receptors in the rat nucleus of the solitary tract and dorsal motor nucleus of the vagus in vitro

1 Recent studies have shown antagonists at the NK1 subtype of receptor for tachykinins are anti‐emetics and suggested that this may result from blockade of tachykinin‐mediated synaptic transmission at a central site in the emetic reflex. 2 We have used intracellular recording in vitro to study the pharmacology of tachykinins in the nucleus of the solitary tract (NST) and dorsal motor nucleus of the vagus (DMNV). 3 Neurones in the NST were depolarized by substance P (SP), the presumed endogenous ligand for the NK1 receptor and these effects were mimicked by the NK1 agonists, SP‐O‐methylester (SPOMe), GR73632 and septide; however, SP was nearly an order of magnitude less potent than the latter two agonists. 4 In the DMNV, SP and NK1 receptor agonists evoked similar depolarising responses but SP appeared to be more potent than in the NST and was closer in potency to the other agonists. 5 NK1‐receptor antagonists blocked responses to septide and GR73632 in the NST but had little effect on responses to SP and SPOMe. In contrast, in the DMNV the NK1‐receptor antagonists blocked responses to septide and GR73632 but also reduced responses to SP and SPOMe. 6 Neurokinin A (NKA) was almost equipotent with septide and GR73632 in depolarizing both NST and DMNV neurones but these effects were not mimicked by a specific NK2‐receptor agonist. Responses to NKA were unaffected by an NK2‐receptor antagonist; however, the depolarizing effects of NKA were blocked by NK1‐receptor antagonists. 7 Neurones in both DMNV and NST were unaffected by the endogenous NK3‐receptor ligand, neurokinin B and by a specific agonist for this site, senktide. 8 The results with NK1 receptor agonists and antagonists suggest that the septide‐sensitive NK1 site is involved in the excitation of both NST and DMNV neurones. The ‘classical’ NK1 receptor may play more of a role in the DMNV and a third unknown site may be responsible for the depolarizing response to SP in the NST. The effects of NKA are best interpreted as an action at the septide‐sensitive NK1 site. This raises the possibility that anti‐emetic action of the NK1 antagonists may be due to blockade of NKA transmission at the septide‐sensitive site.