Claudio Munari

Inhibitory circuits in human dysplastic tissue

Summary: Purpose: Different types of epilepsies and seizures depend on the nature and location of the primary disturbance and are presumably mediated by different physiopathological mechanisms. We immunocytochemically investigated possible changes in the inhibitory ‐aminobutyric acid (GABA)ergic system in specimens taken from four patients who underwent surgery for intractable epilepsy and presented two different types of focal cortical dysplasia in the temporal lobe.

Taylor's cortical dysplasia: a confocal and ultrastructural immunohistochemical study.

In the present report we describe the neuropathological characteristics of tissue surgically resected from three patients affected by intractable epilepsy secondary to cortical dysplasia. Common features, suggestive of a focal cortical dysplasia of Taylor, were observed in all specimens. Immunocytochemical procedures were performed using neuronal and glial markers and the sections were observed at light traditional and confocal microscopes. This part of the investigation pointed out: 1. cortical laminar disruption; 2. very large neurons displaying a pyramidal or round shape; 3. ballooned cells; 4. decrease of calcium binding proteins immunoreactivity; 5. abnormal nets of parvalbumin‐ and glutamic acid decarboxylase‐positive puncta around giant neurons but not around ballooned cells. Ultrastructural investigation on the same material provided evidence of a high concentration of neurofilaments in giant neurons and of glial intermediate filaments in ballooned cells. In addition, immunolabeled GABAergic terminals clustered around giant neurons were not found to establish synapses on their cell bodies.

Surgery for drug resistant partial epilepsy in children with focal cortical dysplasia: anatomical–clinical correlations and neurophysiological data in 10 patients

Objective:To analyse a population of children with focal cortical dysplasia operated on for drug resistant partial epilepsy, with emphasis on clinical features, seizure semiology, interictal and ictal EEG and stereo EEG findings, histological and topographical characteristics of the lesions, extension and localisation of cerebral excision, and its postoperative effect on seizure frequency. Methods:10 patients were studied, aged between 26 months and 11 years (median 6 years). Magnetic resonance imaging (MRI) abnormalities were unilobar (temporal 3, frontal 2), bilobar (2), or multilobar (1); the two patients with negative MRI suffered from frontal seizures. Presurgical diagnostic steps varied in complexity and invasiveness depending on the anatomical/electrical/clinical features of each patient. In four patients they included only scalp video EEG monitoring, and in six, also invasive recordings using stereotactically implanted intracerebral electrodes. Surgery consisted of corticectomy plus lesionectomy in all cases. Results:70% of the patients were seizure-free after a minimum postoperative follow up of 25 months. These included three patients with temporal lesions and four of seven patients with other lobar or multilobar extratemporal localisation. One patient had improvement in seizure control. Outcome was poor in multilobar patients, but a class Ia outcome was obtained in one case after partial lesionectomy associated with bilobar corticectomy. All patients showed developmental improvement. Conclusions:Analysis of the data in these patients allowed the production of an “anatomical-clinical concordance” list, which appeared to be correlated with the diagnostic steps performed. Carrying out a stereo EEG exploration in the most complex cases proved useful in defining the epileptogenic zone in extratemporal and multilobar epilepsies. Stereo EEG recordings facilitated a tailored resection of extralesional cortex.

Intracerebral Low Frequency Electrical Stimulation: a New Tool for the Definition of the “Epileptogenic Area”?

Low Frequency (1 Hz) Electrical Stimulation (LFES) has been systematically utilized, during stereo-EEG investigations, in 24 consecutive young adult patients considered for surgical treatment of severe drug-resistant partial epilepsy. Ninety seizures (1-14/patient) identical to the spontaneous ones previously recorded were thus obtained in 19 patients (79%). LFES is less effective for induction of seizures than high frequency (50 Hz) stimulation (5.9% vs 22.9%), and it also provokes less false positive responses (1% vs 17%). The main sensitive structures to LFES are the hippocampus, the amygdala, and the hippocampal gyrus. However, seizures were also induced by stimulating the temporal lobe white matter, the temporal pole, and the temporal neocortex, as well as the orbito-frontal cortex (in the only patient with fronto-temporal epilepsy). The more frequently observed electrical pattern is a gradual increase of spikes and spikes and waves frequency, with or without occurrence of low voltage fast activity. The high percentage of early subjective manifestations similar to the spontaneous ones, the lack of major electrical artifact, and the good visualization of the spatial evolution of the induced-discharge, strongly suggest that LFES is of great help for defining the epileptogenic area.

Selective block of rat and human neocortex GABAB receptors regulating somatostatin release by a GABAB antagonist endowed with cognition enhancing activity

Previously, we have shown that presynaptic GABA(B) receptors regulating the release of various transmitters from CNS terminals can be differentially blocked by GABA(B) antagonists suggesting the existence of pharmacologically distinct GABA(B) receptor subtypes. We here examined the ability of CGP 36742 [(3-aminopropyl)n-butylphosphinic acid], a selective GABA(B) antagonist endowed with cognition enhancing activity, to block release-regulating GABA(B) receptors. In particular, CGP 36742 was tested against the inhibition of the depolarization-evoked release of GABA, glutamate, cholecystokinin and somatostatin produced by (-)baclofen in rat and human neocortex axon terminals. CGP 36742 potently antagonized (IC50 = 0.14 microM) the inhibition by (-)baclofen of somatostatin release from superfused rat neocortex synaptosomes. In contrast, the effects of (-)baclofen on GABA, glutamate and cholecystokinin release were insensitive to CGP 36742, at concentrations of up to 100 microM. In human neocortex synaptosomes CGP 36742 exhibited a pattern of selectivity identical to that in rat synaptosomes, although the antagonist was at least 10-fold less potent in human than in rat brain. CGP 36742 is the first compound displaying great selectivity for the GABA(B) presynaptic receptors regulating somatostatin release. Considering the proposed implication of the neuropeptide in cognitive processes, disinhibition of somatostatin release merits consideration as one of the mechanisms possibly involved in the behavioral activity of CGP 36742.

Pharmacological diversity between native human 5‐HT1B and 5‐HT1D receptors sited on different neurons and involved in different functions

The releases of [3H]5‐hydroxytryptamine ([3H]5‐HT) and of endogenous glutamic acid and their modulation through presynaptic h5‐HT1B autoreceptors and h5‐HT1D heteroreceptors have been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery. The inhibition by 5‐HT of the K+ (15u2003mM)‐evoked overflow of [3H]5‐HT was antagonized by the 5‐HT1B/5‐HT1D receptor ligand GR 127935, which was ineffective on its own; this drug was previously found to behave as a full agonist at the h5‐HT1D heteroreceptor regulating glutamate release. The recently proposed selective h5‐HT1B receptor ligand SB‐224289 also prevented the effect of 5‐HT at the autoreceptor, being inactive on its own; in contrast, SB‐224289, at 1u2003μM, was unable to interact with the h5‐HT1D heteroreceptor. The inhibitory effect of 5‐HT on the K+‐evoked overflow of glutamate was antagonized by the h5‐HT1D receptor ligand BRL‐15572; added in the absence of 5‐HT the compound was without effect. BRL‐15572 (1u2003μM) was unable to modify the effect of 5‐HT at the autoreceptor regulating [3H]5‐HT release. The selective 5‐HT1A receptor antagonist (+)‐WAY 100135, previously found to be an agonist at the h5‐HT1D heteroreceptor regulating glutamate release, could not interact with the h5‐HT1B autoreceptor when added at 1u2003μM. It is concluded that native h5‐HT1B and h5‐HT1D receptors exhibit a hitherto unexpected pharmacological diversity.

Native human neocortex release-regulating dopamine D2 type autoreceptors are dopamine D2 subtype.

Dopamine (DA) autoreceptors expressed at DA nerve terminals regulate DA release. Considerable evidence has indicated that, in rodents, these autoreceptors belong to the D2 type of the DA receptor family, which, in turn, comprises the D2, D3 and D4 subtypes. We investigated here, for the first time, the subclassification of native human DA autoreceptors by studying the release of [3H]DA evoked by electrical stimulation in fresh human neocortical slices. The results have been compared with those obtained in three animal systems: rat neocortical and striatal slices and rat mesencephalic neuronal cultures. In human neocortical slices, the D2/D3 receptor agonist quinpirole (1u2003n m–10u2003μm) inhibited tritium release with a calculated EC50 of 17u2003n m and a maximal inhibition of ≈u200375% reached at 1u2003μm. In the presence of the D2/D3 receptor antagonist (–)‐sulpiride (0.1 and 1u2003μm), the concentration–response curve of quinpirole was shifted to the right, and the apparent pA2 mean value was 8.5 (8.14–8.77); on the other hand, the inhibitory effects of quinpirole were not affected by the D3 receptor‐selective antagonist [7‐N,N‐dipropylamino‐5,6,7,8‐tetrahydro‐naphtho(2,3b) dihydro,2,3‐furane] (S 14297) and the D4 receptor‐selective antagonist 3‐(4‐[4‐chlorophenyl]piperazin‐1‐yl)‐methyl‐1H‐pyrrolo [2,3‐b]pyridine (L‐745,870) (0.01–1u2003μm in each case). Superimposable results have been obtained when the release was elicited from rat striatal slices or dopamine mesencephalic neurons in culture, whereas quantitative differences emerged in the case of rat cortical slices. It is concluded that in human brain, as well as in rat brain, the release of DA in the terminal region of midbrain dopaminergic neurons is regulated through autoreceptors of the D2 subtype.

Clinical Ictal Symptomatology and Anatomical Lesions: Their Relationships in Severe Partial Epilepsy

Summary High‐resolution imaging techniques can demonstrate anatomic alterations in most patients identified as candidates for surgical treatment of their partial epilepsy. The demonstration of an anatomic lesion is only one step in the presurgical diagnostic procedure, which includes video‐EEG and, when necessary, video‐stereo‐EEG recordings of seizures. A review of the literature shows that the simple removal of the magnetic resonance imaging (MRI)‐evident lesion (“lesionectomy”) reduces but does not completely suppress seizures in a large percentage of patients, especially those with neuronal migration disorders. This phenomenon could, at least in part, be explained by preliminary data (in 33 patients) showing that less than 20% of seizures correspond to a well‐localized, intra‐lesional discharge in about 40% of stereo‐EEG‐investigated patients with at least one intralesional electrode. The authors illustrate some anatomo‐electroclinical examples of intra‐individual variability of the ictal symptomatology, raising the problem of the decision about the extent of the surgical removal. Recent histologic and immunohistochemical studies have demonstrated several kinds of structural alterations in the stereo‐EEG‐defined epileptogenic zone, not always overlapping with the MRI‐visible lesion. This aspect can further explain some failures of MRI‐guided lesionectomies. That relationships between “lesions” and epileptogenic zones may be variable is also suggested by reports of patients who present with multiple lesions (i.e., cavernous angiomas, Bourneville syndrome) and are cured by removal of only one of them.

Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage‐sensitive calcium channels

1 Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L‐[3H]‐arginine to L‐[3H]‐citrulline. 2 Elevation of extracellular K+ to 20, 35 or 60u2003mM concentration‐dependently augmented L‐[3H]‐citrulline production. The response to 35u2003mM KCl was abolished by NG‐nitro‐L‐arginine (100u2003μM) demonstrating NO synthase specific conversion of L‐arginine to L‐citrulline. Increasing extracellular MgCl2 concentration up to 10u2003mM also prevented the K+ (35u2003mM)‐induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity. 3 However, the effect of high K+ (35u2003mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK‐801), 6‐nitro‐7‐sulphamoylbenzo(f)quinoxaline‐2–3‐dione (NBQX) or L‐2‐amino‐3‐phosphonopropionic acid (L‐AP3) as well as to the nicotinic receptor antagonist, mecamylamine. 4 The 35u2003mM K+ response was insensitive to ω‐conotoxin GVIA (1u2003μM) and nifedipine (100u2003μM), but could be prevented in part by ω‐agatoxin IVA (0.1 and 1u2003μM). The inhibition caused by 0.1u2003μM ω‐agatoxin IVA (∼30%) was enhanced by adding ω‐conotoxin GVIA (1u2003μM) or nifedipine (100u2003μM). Further inhibition (up to above 70%) could be observed when the three Ca2+ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100u2003μM) the K+‐evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1u2003μM ω‐conotoxin GVIA plus 100u2003μM nifedipine. No further inhibition could be observed upon addition of MK‐801 or/and NBQX. 5 It was concluded that elevation of extracellular [K+] causes NO synthase activation by external Ca+ entering cells mainly through channels of the P/Q‐type. Other Ca2+ channels (L‐ and N‐type) appear to contribute when P/Q‐channels are blocked.

Biopsie seriate stereotassiche e correlazioni con TC ed RM nei gliomi a lento accrescimento

La necessita di accertare mediante biopsie stereotattiche la natura di una lesione endocranica per modulare lapproccio terapeutico e stata avvertita per primi da Talairach e coiL, sia nei tumori ipofisari 25 che nei gliomi 26• Questa esigenza era apparsa talmente naturale agli Autori, che linformazione dellavvenuto prelievo stereotattico e relegata in fondo all articolo. E interessante ricordare che la metodologia stereotattica e stata usata prima per cercare di curare 13 , che per cercare di ottenere una diagnosi. I primi lavori che descrivono dettagliatamente la metodologia delle biopsie stereotattiche risalgono agli anni 60 10• V a anche ricordato che i neurochirurghi «classici» criticavano aspramente una metodologia che ai loro occhi, non avvezzi ad una visione tridimensionale del cervello, appariva pericolosa, soprattutto perche considerata, a torto, «cieca» 26 • Il primo «computer» nasce un anno prima della chirurgia stereotattica: Kall nel 1987 12 nota, con umorismo, che le due vite hanno avuto unevoluzione parallela fino allavvento della TC · Sono di questo periodo le prime grandi serie che riportano i risultati delle biopsie stereotattiche 4•21 • L avvento della Risonanza Magnetica aveva suscitato la speranza di poter evitare un accertamento diagnostico invasivo grazie al miglioramento delle tecniche di «imagerie». Molto rapidamente si e capito che il vecchio problema di poter distinguere uninfiltrazione tumorale dalledema 7, restava sostanzialmente irrisolto 7 . Un problema particolare e rappresentato dallesigenza di una diagnosi preoperatoria in pazienti con una alterazione morfologica cerebrale (TC RM) ed unepilessia parziale grave farmacoresistente 15 , soprattutto nei bambini 18, sapendo che, se la maggioranza delle lesioni e tumorale 5 le eccezioni sono lungi dall essere rare 19• In questa breve nota cercheremo di valutare il contributo delle biopsie stereotattiche alla diagnosi differenziale nei Gliomi a lento accrescimento, spesso solo grossolanamente suggerita dal quadro clinico, dalla TC e dalla RM.