Thalamic reticular nucleus impairments and abnormal prefrontal control of dopamine system in a developmental model of schizophrenia: prevention by N-acetylcysteine.

Recent evidence showed thalamic abnormalities in schizophrenia involving disruptions to the parvalbumin neurons in the thalamic reticular nucleus (TRN). However, their functional consequences, as well as a potential linkage to oxidative stress, are unclear. The TRN is posited to gate prefrontal control of dopamine neuron activity in the ventral tegmental area (VTA). Thus, we hypothesized that schizophrenia-related TRN abnormalities might contribute to dopamine dysregulation, a well-known feature of the disorder. To test this, in adult rats exposed prenatally to methylazoxymethanol acetate (MAM rats), oxidative impairments to the parvalbumin neurons in the anterior TRN were assessed by immunohistochemistry. Using in vivo electrophysiology, we investigated whether inactivation of the prefrontal cortex would produce differential effects on VTA dopamine neurons in MAM rats. We show that MAM rats displayed reduced markers of parvalbumin and wisteria floribunda agglutinin-labeled perineuronal nets, correlating with increased markers of oxidative stress (8-oxo-7, 8-dihydro-20-deoxyguanosine, and 3-nitrotyrosine). Moreover, MAM rats displayed heightened baseline and abnormal prefrontal control of VTA dopamine neuron activity, as tetrodotoxin-induced inactivation of the infralimbic prefrontal cortex decreased the dopamine population activity, contrary to the normal increase in controls. Such dopamine neuron dysregulation was recapitulated by enzymatic perineuronal net digestion in the TRN of normal rats. Furthermore, juvenile (postnatal day 11-25) antioxidant treatment (N-acetyl-cysteine, 900 mg/L drinking water) prevented all these impairments in MAM rats. Our findings suggest that early accumulation of oxidative stress in the TRN may shape the later onset of schizophrenia pathophysiology, highlighting redox regulation as a potential target for early intervention.

Reduced risk aversion and impaired short-term memory in juvenile rats with malformation of cortical development.

Malformation of cortical developments (MCDs) is currently an incurable disease and is associated with significant neuropsychological problems, such as intellectual disability, epilepsy, and anxiety disorders from a young age. Development of a suitable animal model and pathophysiological study is therefore necessary to better understand and treat MCDs from being an incurable disease. The Y-maze, open field, and fear conditioning studies were performed at postnatal days 40-44 to validate the behavioral phenotypes of the existing rat model of MCD with prenatal methylazoxymethanol exposure at their developmental period. The study results show that juvenile rats with MCD spent significantly less time inside the novel arms in Y-maze and less time in the peripheral zones of the open field. Additionally, the rats with MCDs showed attenuated freezing behavior to sound and light cues as well as to context after fear conditioning. This comprehensive behavioral analysis of rats with MCDs at the juvenile period indicate a lack of spatial memory, decreased anxiety, and learning disability in these rats, which is compatible with the human behavioral phenotype of MCDs and can be used as the behavioral biomarkers for future translational research.

A Network Analysis of Epigenetic and Transcriptional Regulation in a Neurodevelopmental Rat Model of Schizophrenia With Implications for Translational Research.

Prenatal administration of mitotoxin methylazoxymethanol acetate (MAM) in rats produces behavioral, pharmacological, and anatomical abnormalities once offspring reach adulthood, thus establishing a widely used neurodevelopmental model of schizophrenia. However, the molecular aspects underlying this disease model are not well understood. Therefore, this study examines epigenetic and transcriptional dysregulation in the prefrontal cortex and hippocampus of MAM rats as these are brain regions closely associated with schizophrenia pathogenesis. Upon sequencing messenger and microRNA (mRNA and miRNA, respectively), differential expression was revealed in the prefrontal cortex and hippocampus between MAM- and saline-treated rats; sequencing data were validated by qualitative real-time polymerase chain reaction. Bioinformatic analyses demonstrated that the differentially expressed (DE) genes were strongly enriched in interactive pathways related to schizophrenia, including chemical synaptic transmission, cognition, and inflammatory responses; also, the potential target genes of the DE miRNAs were enriched in pathways related to synapses and inflammation. The blood of schizophrenia patients and healthy controls was further analyzed for several top DE mRNAs: DOPA decarboxylase, ret proto-oncogene, Fc receptor-like 2, interferon lambda receptor 1, and myxovirus (influenza virus) resistance 2. The results demonstrated that the expression of these genes was dysregulated in patients with schizophrenia; combining these mRNAs sufficiently differentiated schizophrenia patients from controls. Taken together, this study suggests that the MAM model has the potential to reproduce hippocampus and prefrontal cortex abnormalities, relevant to schizophrenia, at the epigenetic and transcriptional levels. These data also provide novel targets for schizophrenia diagnoses and treatments.

Effect of ablated hippocampal neurogenesis on the formation and extinction of contextual fear memory.

Newborn neurons in the subgranular zone (SGZ) of the hippocampus incorporate into the dentate gyrus and mature. Numerous studies have focused on hippocampal neurogenesis because of its importance in learning and memory. However, it is largely unknown whether hippocampal neurogenesis is involved in memory extinction per se. Here, we sought to examine the possibility that hippocampal neurogenesis may play a critical role in the formation and extinction of hippocampus-dependent contextual fear memory. By methylazoxymethanol acetate (MAM) or gamma-ray irradiation, hippocampal neurogenesis was impaired in adult mice. Under our experimental conditions, only a severe impairment of hippocampal neurogenesis inhibited the formation of contextual fear memory. However, the extinction of contextual fear memory was not affected. These results suggest that although adult newborn neurons contribute to contextual fear memory, they may not be involved in the extinction or erasure of hippocampus-dependent contextual fear memory.

Combined effects of prenatal inhibition of vasculogenesis and neurogenesis on rat brain development.

Malformations of cortical development (MCD) are one of the most common causes of neurological disabilities including autism and epilepsy. To disrupt cortical formation, methylazoxymethanol (MAM) or thalidomide (THAL) has been used to affect neurogenesis or vasculogenesis. Although previous models of MCD have been useful, these models primarily attack a single aspect of cortical development. We hypothesized that simultaneous prenatal exposure to MAM or THAL will lead to the development of a novel and specific type of brain maldevelopment. Rats were prenatally exposed to MAM and THAL. At early postnatal days, brains displayed abnormal ventricular size and hemispheric asymmetry due to altered brain water homeostasis. The postnatal brain was also characterized by gliosis in regions of focal leakage of the blood brain barrier. These morphological abnormalities gradually disappeared at adult stages. Although the adult MAM-THAL rats showed normal cortical morphology, abnormal hippocampal connectivity and mossy fiber sprouting persisted well into adulthood.

Reelin is essential for neuronal migration but not for radial glial elongation in neonatal ferret cortex.

Numerous functions related to neuronal migration are linked to the glycoprotein reelin. Reelin also elongates radial glia, which are disrupted in mutant reeler mice. Our lab developed a model of cortical dysplasia in ferrets that shares features with the reeler mouse, including impaired migration of neurons into the cerebral cortex and disrupted radial glia. Explants of normal ferret cortex in coculture with dysplastic ferret cortex restore the deficits in this model. To determine if reelin is integral to the repair, we used explants of P0 mouse cortex either of the wild type (WT) or heterozygous (het) for the reelin gene, as well as P0 reeler cortex (not containing reelin), in coculture with organotypic cultures of dysplastic ferret cortex. This arrangement revealed that all types of mouse cortical explants (WT, het, reeler) elongated radial glia in ferret cortical dysplasia, indicating that reelin is not required for proper radial glial morphology. Migration of cells into ferret neocortex, however, did not improve with explants of reeler cortex, but was almost normal after pairing with WT or het explants. We also placed an exogenous source of reelin in ferret cultures at the pial surface to reveal that migrating cells move toward the reelin source in dysplastic cortex; radial glia in these cultures were also improved toward normal. Our results demonstrate that the normotopic position of reelin is important for proper neuronal positioning, and that reelin is capable of elongating radial glial cells but is not the only radialization factor.

Altered glutamate receptor - transporter expression and spontaneous seizures in rats exposed to methylazoxymethanol in utero.

PURPOSE: Brain malformations are a common cause of intractable epilepsy and cognitive dysfunction in children. Prenatal exposure to the teratogen methylazoxymethanol (MAM) is a rodent model of brain malformation featuring loss of lamination, clusters of displaced hippocampal cells, and pharmaco-resistance to antiepileptic drugs. In a normotopic hippocampus, expression of postsynaptic glutamate receptors and the transporters regulating neurotransmitter reuptake are critical factors modulating excitation and synaptic communication. Alterations in this system can have profound effects on overall excitability, cognitive function, and seizure thresholds. METHODS: Immunohistochemical techniques were used to analyze the expression of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5 methylisoxazole-4-proprionic acid (AMPA) receptor subunits in rats exposed to MAM in utero (25 mg/kg, intraperitoneal injection). We also examined the expression of several glutamate transporters (EAAC1, vGLUT1, and vGLUT2). A video-electroencephalographic (video-EEG) system was used for long-term monitoring of adult MAM-exposed rats. RESULTS: Heterotopic hippocampal neurons exhibited striking reductions in GluR1 and EAAC1 expression; vGlut2 expression was prominent in these regions. Spontaneous electrographic seizures were verified in two animals. CONCLUSIONS: We conclude that glutamate receptor subunit and transporter expression are altered in animals exposed to MAM in utero. Further studies in the MAM model may provide greater insight into the potential disruptions in excitatory synaptic neurotransmission that can occur in a malformed brain.

Exposure in fetus of methylazoxymethanol in the rat alters brain neurotrophins' levels and brain cells' proliferation.

Changes during gestation have been shown to induce brain maldevelopment associated with changes in neurotrophins as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neuropsychiatric disorders in humans. A rat model of altered prenatal brain development resembling the onset of schizophrenia has been obtained by administering in fetus methylazoxymethanol (MAM) at gestational day 12 which impairs the growth of limbic pathways between the entorhinal cortex and the hippocampus. Using the MAM model we studied in young rats the brain levels of both NGF/BDNF and their main receptors, TrkA/TrkB, to investigate whether or not changes in neurotrophins could affect the presence of brain BrdU positive cells. We found increased NGF and BDNF protein levels, associated with elevated TrkA and TrkB expression, in the hippocampus, entorhinal cortex, olfactory lobes and subventricular zone (SVZ), brain areas playing a key role in the production and migration of new dividing cells. We also found higher levels of BrdU positive cells in the SVZ and hippocampus but not a significant potentiation in the entorhinal cortex and olfactory lobes. All together the findings indicate that prenatal MAM exposure in young rats may elicit both neurotrophins' elevation and cell proliferation in limbic brain areas.

Stromal-derived factor-1 (CXCL12) regulates laminar position of Cajal-Retzius cells in normal and dysplastic brains.

Normal brain development requires a series of highly complex and interrelated steps. This process presents many opportunities for errors to occur, which could result in developmental defects in the brain, clinically referred to as malformations of cortical development. The marginal zone and Cajal-Retzius cells are key players in cortical development and are established early, yet there is little understanding of the factors resulting in the disruption of the marginal zone in many types of cortical malformation syndromes. We showed previously that treatment with methylazoxymethanol in rats causes marginal zone dysplasia with displacement of Cajal-Retzius cells to deeper cortical layers. Here we establish that loss of activity of the chemokine stromal-derived factor-1 (SDF1) (CXCL12), which is expressed by the leptomeninges, is necessary and sufficient to cause marginal zone disorganization in this widely used teratogenic animal model. We also found that mice with mutations in the main receptor for SDF1 (CXCR4) have Cajal-Retzius cells displaced to deeper cortical layers. Furthermore, by inhibiting SDF1 signaling in utero by intraventricular injection of a receptor antagonist, we establish that SDF1 signaling is required for the maintenance of Cajal-Retzius cell position in the marginal zone during normal cortical development. Our data imply that cortical layering is not a static process, but rather requires input from locally produced molecular cues for maintenance, and that complex syndromes of cortical malformation as a result of environmental insults may still be amenable to explanation by interruption of specific molecular signaling pathways.

Factors affecting the morphology of radial glia.

A model of cortical dysplasia results from disruption of the earliest generated neocortical cells. Injections of an antimitotic (methylazoxy methanol - MAM) into pregnant ferrets result in a constellation of effects, which include disruption of radial glia, with early differentiation in astrocytes, and impaired migration of neurons into the cortical plate. We found previously that culture of P0 MAM-treated slices with explants of normal cortical plate reorganizes the radial glia toward their normal morphology and improves migration of neurons into the cortical plate. This suggested that P0 normal cortical plate contains a 'factor' capable of providing reorganizing cues to disorganized developing cortex. The current study characterizes the biological activity in normal cortical plate by isolating fractions of different molecular weight obtained from conditioned media of organotypic cultures. The only media fraction capable of providing reorganizing activity to MAM-treated cortex was the molecular weight fraction between 30 and 50 kDa. Treatment designed to denature proteins demonstrated that the active molecular weight fraction (30-50 kDa) was not able to provide reorganizing cues when either heated or treated with Proteinase K. These data provide support for the idea that normal cortical plate of neonatal ferret contains a radialization factor that is a protein of 30-50 kDa.

Developmental expression of the cell cycle and apoptosis controlling gene, Lot1, in the rat cerebellum and in cultures of cerebellar granule cells.

The Lot1 gene encodes a zinc finger protein that, in vitro, concurrently regulates apoptosis and cell cycle arrest and belongs to a recently identified family of proteins with oncogenic and tumor-supressor functions. The present study, based on the development of the first antibody reportedly produced against rat Lot1, examines protein expression during normal development of the rat cerebellum and following methylazoxymethanol (MAM) administration, which results in hypoplasia of the cerebellar granule cell population. Using light microscopic immunocytochemistry, specific immunostaining for the Lot1 protein was observed at postnatal days 2 to 7 in the superficial external granule layer composed primarily of proliferating neuronal precursor cells. Purkinje cells showed distinct nuclear labeling at P7. In the adult cerebellum, the overall low Lot1 level was essentially associated with Purkinje cells. Experimentally altered developmental conditions, such as those obtained through MAM-induced microencephaly, did not drastically affect the pattern of Lot1 expression. In particular, Purkinje cells continued to show normal levels of immunoreactivity notwithstanding the altered cerebellar architecture. Primary cultures of cerebellar granule cells showed a temporal pattern of Lot1 expression resembling that of in vivo development, with mRNA and protein levels progressively decreasing with differentiation. When cerebellar granule cells were exposed to different neurotoxic challenges, Lot1 appeared not affected by purely apoptotic cell death, while transitorily induced by mixed necrotic-apoptotic cell death.

An examination of calcium current function on heterotopic neurons in hippocampal slices from rats exposed to methylazoxymethanol.

PURPOSE: To study voltage-dependent calcium currents (VDCCs) on hippocampal heterotopic neurons by using whole-cell patch-clamp techniques in brain slices prepared from methylaxozymethanol (MAM)-exposed rats. METHODS: Whole-cell voltage-clamp recordings were obtained from visually identified neurons in acute brain slices by using an infrared differential interference contrast (IR-DIC) video microscopy system. Heterotopic neurons were compared with normotopic pyramidal cells in hippocampal slices from MAM-exposed rats or CA1 pyramidal neurons in slices from controls. RESULTS: Heterotopic neurons expressed a prominent VDCC, which exhibited a peak current maximum around -30 mV (holding potential, -60 mV) and an inactivation time constant of 48.2 +/- 2.4 ms (n = 91). VDCC peak current and inactivation time constants were similar for normotopic (n = 92) and CA1 pyramidal cells (n = 40). Pharmacologic analysis of VDCC, on heterotopic, normotopic, and CA1 pyramidal cells, revealed an approximately 70% blockade of peak Ca2+ current with nifedipine and amiloride (L- and T-type channel blockers, respectively). Inhibition of VDCC, for all three cell types, also was similar when more specific Ca2+ channel antagonists were used [e.g., omega-conotoxin GVIA (N-type), omega-agatoxin KT (P/Q-type), and sFTX-3.3 (P-type)]. VDCC modulation by norepinephrine (NE) or adrenergic receptor-specific agonists [clonidine (alpha2), isoproterenol (beta), and phenylephrine (alpha1)] was similar for heterotopic and CA1 pyramidal cells. CONCLUSIONS: Heterotopic neurons do not appear to exhibit Ca2+ channel abnormalities that could contribute to the reported hyperexcitability associated with MAM-exposed rats.

Comparative potencies of induction of point mutations and genetic duplications by the methylating agents methylazoxymethanol and dimethyl sulfate in bacteria.

Methylazoxymethanol (MAM) and dimethyl sulfate (DMS) are mutagens whose genetic effects can be ascribed to the methylation of DNA. While both methylate the N7 position of guanine heavily, only MAM strongly methylates the O(6) position of guanine. We evaluated the relative effectiveness and specificity of MAM and DMS in bacterial assays for the induction of point mutations and the formation of chromosomal duplications by genetic recombination. Salmonella typhimurium strain TS1121 was used to measure the formation of genetic duplications on the basis of the aroC321 allele and mutations by reversion of the hisG46 allele. Specific base pair substitutions and frameshift mutations were measured in a reversion assay based on lacZ alleles of Escherichia coli. The results show MAM to be the more potent mutagen and DMS the stronger recombinagen in the Salmonella assay. In the lacZ assay DMS induced several classes of base pair substitutions (GC-->AT transitions, GC-->TA transversions and AT-->TA transversions), as well as lower frequencies of +1, -1 and -2 frameshift mutations. The activity of MAM as a base pair substitution mutagen was more specific than that of DMS, inducing only GC-->AT transitions. It also induced +G, -G, -A and -CG frameshift mutations, though more weakly than it induced GC-->AT transitions. Long known as a base pair substitution mutagen, the induction of frameshifts by MAM was unexpected. The results show that both DMS and MAM are effective inducers of base pair substitutions and modest inducers of frameshifts and that DMS exhibits a broader spectrum of mutagenic activity than does MAM.

Postnatal development of rat dentate gyrus: effects of methylazoxymethanol administration.

In order to investigate the role of postnatal neurogenesis in granule cell number control in the rat dentate gyrus, we administered Methylazoxymethanol (MAM), a drug able to prevent cells from dividing, on P3, P5, P7, P9, when the most granule cells are produced. The effect of MAM on the number of proliferating precursors and of granule cells was examined at P16 and P90. We used 5-bromo-2'-deoxyuridine administration to label proliferating cells and immunohistochemistry to characterize the cell phenotype using neuron markers TUC 4, PSA-NCAM, Calbindin D28K and glial marker GFAP. At 16 days of age in MAM-treated rats we observed a significant decrease of BrdU-positive cells. Consistently, a decrease in density and number of granule cells was found compared to the controls. At 90 days the dentate gyrus of treated rats showed a complete recovery: no differences in the density, total number of neurons, the BrdU- and TUC 4-positive cells were revealed with respect to the controls. No deficits were evident in performance on the water maze in MAM-treated rats. These data suggest that the dentate gyrus is able to re-establish the proliferative zone and to rebuild the granule cell layer following neonatal MAM administration.

Methylazoximethanol acetate-induced cell death in the granule cell layer of the developing mouse cerebellum is associated with caspase-3 activation, but does not depend on the tissue-type plasminogen activator.

Methylazoximethanol (MAM) acetate-induced cell death in the external granule cell layer of the developing cerebellum affects clusters of cells with morphological features of apoptosis. This is accompanied by selective induction of active caspase-3 expression and increased c-Jun/AP-1 (N) immunoreactivity in dying cells, as revealed with immunohistochemistry. Since the antibody to cJun/AP-1 (N) cross-reacts with epitopes emerging after caspase-mediated proteolysis during apoptosis, these results indicate that MAM-induced cell death is associated with active caspase-3 expression and function in dying cells. In order to investigate the involvement of tissue-type plasminogen activator (tPA), which has been implicated in certain forms of neuronal cell death, MAM-induced cell death has been examined in tPA-/- and tPA+/+ mice. No differences in the number of dying cells, as seen with haematoxylin and eosin staining and in situ end-labelling of fragmented nuclear DNA-processed sections, were seen between tPA-/- and tPA+/+ mice. These results indicate that tPA is not involved in MAM-induced cell death in the developing brain.

Constitutive overactivation of protein kinase C in guinea pig brain increases alpha-secretory APP processing without decreasing beta-amyloid generation.

Whilst it is generally accepted that the activation of protein kinase C (PKC) increases amyloid precursor protein (APP) secretion in vitro, the role of PKC in the regulation of APP processing and beta-amyloid generation in vivo is still not well understood. In order to address this question, we established the animal model of neocortical microencephalopathy in guinea pigs caused by in utero treatment with methylazoxymethanol acetate, a DNA-methylating substance that eliminates proliferating cells of neuroepithelial origin. The induction of this neocortical malformation is accompanied by constitutive overactivation of PKC in the neocortex of the offspring. In the cortical and hippocampal tissues of juvenile microencephalic guinea pigs (postnatal day 30), we observed significant increases in basal (by 58% and 74%, respectively,) and phorbol ester-stimulated PKC enzyme activity (by 47% and 71%) as compared to age-matched control animals. In the same cortical/hippocampal preparations of methylazoxymethanol-treated animals, there was increased alpha-secretion of APP by 35% and 30% as measured by Western blot analysis using the antibody 6E10, whilst total APP secretion as well as APP mRNA expression remained unaltered. This upregulation of APP alpha-secretion was limited to brain areas that displayed elevated PKC activity. However, constitutive overactivation of neocortical PKC did not affect the generation of beta-amyloid peptides 1-40 or 1-42 as measured by ELISA, suggesting that only the alpha-secretase pathway of APP processing is affected by chronic PKC overactivation in vivo.

Characterization of heterotopic cell clusters in the hippocampus of rats exposed to methylazoxymethanol in utero.

Cortical disorganization represents one of the major clinical findings in many children with medically intractable epilepsy. To study the relationship between seizure propensity and abnormal cortical structure, we have begun to characterize an animal model exhibiting aberrant neuronal clusters (heterotopia) and disruption of cortical lamination. In this model, exposing rats in utero to the DNA methylating agent methylazoxymethanol acetate (MAM; embryonic day 15) disrupts the sequence of normal brain development. In MAM-exposed rats, cells in hippocampal heterotopia exhibit neuronal morphology and do not stain with immunohistochemical markers for glia. In hippocampal slices from MAM-exposed animals, extracellular field recordings within heterotopia suggest that these dysplastic cell clusters make synaptic connections locally (i.e. within the CA1 hippocampal subregion) and also make aberrant synaptic contact with neocortical cells. Slice perfusion with bicuculline or 4-aminopyridine leads to epileptiform activity in dysplastic cell clusters that can occur independent of input from CA3. Taken together, our findings suggest that neurons within regions of abnormal hippocampal organization are capable of independent epileptiform activity generation, and can project abnormal discharge to a broad area of neocortex, as well as hippocampus.

Bcl-2 and Bax expression following methylazoxymethanol acetate-induced apoptosis in the external granule cell layer of the developing rat cerebellum.

Since Bcl-2 protects a variety of cell types from programmed cell death, whereas Bax promotes apoptosis, the present study examines Bcl-2 and Bax proteins, and bcl-2 and bax mRNA expression in the developing cerebellum of the rat following methylazoxymethanol (MAM) acetate administration by using immunohistochemistry, Western blotting and Northern blotting. Bcl-2 expression in the developing cerebellum is observed in proliferating and differentiating cells, whereas Bax expression is higher in differentiating cells than in proliferating cells during development. Administration of MAM (0.05 microliter/g, i.p.) at postnatal day 3 produces apoptotic cell death, as detected by the characteristic morphology and positivity with the method of in situ end-labeling of nuclear DNA fragmentation of dying cells, in the external granule cell layer of the cerebellum. Dying cells are not stained with Bcl-2 and Bax antibodies. Furthermore, no modification in the intensity of Bcl-2 and Bax protein bands and in the intensity of Bcl-2 and bax mRNA bands on Western and Northern blots, respectively, were observed between control and treated rats. These data indicate that MAM-induced apoptosis is not associated with modifications in the expression of Bcl-2 and Bax.

Dysplastic neocortex and subcortical heterotopias in methylazoxymethanol-treated rats: an intracellular study of identified pyramidal neurones.

Intracellular recordings were obtained using biocytin-filled electrodes from 78 neurones located in both dysplastic neocortex and subcortical heterotopic aggregates in a model of neuronal migration disorder induced in rats by means of a double methylazoxymethanol injection given on embryonic day 15. Both regular spiking and intrinsically bursting pyramidal neurones were found in all of the examined structures and were synaptically activated by subcortical stimulation. In a neuronal subpopulation (22%) located in the neocortex as well as in the subcortical heterotopic aggregates, the injection of depolarising current pulses elicited aberrant firing patterns, consisting of repetitive bursts of APs that gradually increased in duration and eventually merged in a long-lasting discharge. The gradual development of this 'excessive' bursting behaviour suggests a progressive run-down of the slow components of the hyperpolarising afterpotential.