Tetraose steroidal glycoalkaloids from potato provide resistance against Alternaria solani and Colorado potato beetle.

Plants with innate disease and pest resistance can contribute to more sustainable agriculture. Natural defence compounds produced by plants have the potential to provide a general protective effect against pathogens and pests, but they are not a primary target in resistance breeding. Here, we identified a wild relative of potato, Solanum commersonii, that provides us with unique insight in the role of glycoalkaloids in plant immunity. We cloned two atypical resistance genes that provide resistance to Alternaria solani and Colorado potato beetle through the production of tetraose steroidal glycoalkaloids (SGA). Moreover, we provide in vitro evidence to show that these compounds have potential against a range of different (potato pathogenic) fungi. This research links structural variation in SGAs to resistance against potato diseases and pests. Further research on the biosynthesis of plant defence compounds in different tissues, their toxicity, and the mechanisms for detoxification, can aid the effective use of such compounds to improve sustainability of our food production.

Farmers often rely on pesticides to protect their crops from disease and pests. However, these chemicals are harmful to the environment and more sustainable strategies are needed. This is particularly true for a disease known as the early blight of potato, which is primarily treated using fungicides that stop the fungal pathogen responsible for the infection (Alternaria solani) from growing. An alternative approach is to harness the natural defence systems that plants already have in place to protect themselves. Like humans, plants have an immune system which can detect and destroy specific pathogens. On top of this, they release defence compounds that are generally toxic to pests and microbes, stopping them from infiltrating and causing an infection. In 2021, a group of researchers discovered a wild relative of the potato, known as Solanum commersonii, with strong resistance to early blight disease. Here, Wolters et al. ­ including some of the researchers involved in the 2021 study ­ set out to find how this plant defends itself from the fungus A. solani. The team found that two closely linked genes are responsible for the resistant behaviour of S. commersonii, which both encode enzymes known as glycosyltransferases. Further experiments revealed that the enzymes protect S. commersonii from early blight disease by modifying steroidal glycoalkaloids, typical defence compounds found in potato and other plants from the same family. The glycosyltransferases alter glycoalkaloids in S. commersonii by adding a sugar group to a specific part of the compound called glycone. Wolters et al. found that the glycoalkaloids from S. commersonii were able to slow the growth of other fungal pathogens that harm potatoes when tested in the laboratory. They also made plants resistant to another common destroyer of crops, the Colorado potato beetle. These findings could help farmers breed potatoes and other crops that are more resistant to early blight disease and Colorado potato beetle, as well as potentially other fungi and pests. However, further experiments are needed to investigate how these glycone-modified glycoalkaloids affect humans, and how variants of glycoalkaloids are produced and degraded in different parts of the plants. Acquiring this knowledge will help to employ these defence compounds in a safe and effective manner.

Functional diversification of a wild potato immune receptor at its center of origin.

Plant cell surface pattern recognition receptors (PRRs) and intracellular immune receptors cooperate to provide immunity to microbial infection. Both receptor families have coevolved at an accelerated rate, but the evolution and diversification of PRRs is poorly understood. We have isolated potato surface receptor Pep-13 receptor unit (PERU) that senses Pep-13, a conserved immunogenic peptide pattern from plant pathogenic Phytophthora species. PERU, a leucine-rich repeat receptor kinase, is a bona fide PRR that binds Pep-13 and enhances immunity to Phytophthora infestans infection. Diversification in ligand binding specificities of PERU can be traced to sympatric wild tuber-bearing Solanum populations in the Central Andes. Our study reveals the evolution of cell surface immune receptor alleles in wild potato populations that recognize ligand variants not recognized by others.

Whole-genome sequencing elucidates the species-wide diversity and evolution of fungicide resistance in the early blight pathogen Alternaria solani.

Early blight of potato is caused by the fungal pathogen Alternaria solani and is an increasing problem worldwide. The primary strategy to control the disease is applying fungicides such as succinate dehydrogenase inhibitors (SDHI). SDHI-resistant strains, showing reduced sensitivity to treatments, appeared in Germany in 2013, shortly after the introduction of SDHIs. Two primary mutations in the SDH complex (SdhB-H278Y and SdhC-H134R) have been frequently found throughout Europe. How these resistances arose and spread, and whether they are linked to other genomic features, remains unknown. For this project, we performed whole-genome sequencing for 48 A. solani isolates from potato fields across Europe to better characterize the pathogen's genetic diversity in general and understand the development and spread of the genetic mutations that lead to SDHI resistance. The isolates can be grouped into seven genotypes. These genotypes do not show a geographical pattern but appear spread throughout Europe. We found clear evidence for recombination on the genome, and the observed admixtures might indicate a higher adaptive potential of the fungus than previously thought. Yet, we cannot link the observed recombination events to different Sdh mutations. The same Sdh mutations appear in different, non-admixed genetic backgrounds; therefore, we conclude they arose independently. Our research gives insights into the genetic diversity of A. solani on a genome level. The mixed occurrence of different genotypes, apparent admixture in the populations, and evidence for recombination indicate higher genomic complexity than anticipated. The conclusion that SDHI tolerance arose multiple times independently has important implications for future fungicide resistance management strategies. These should not solely focus on preventing the spread of isolates between locations but also on limiting population size and the selective pressure posed by fungicides in a given field to avoid the rise of new mutations in other genetic backgrounds.

Qualitative and Quantitative Resistance against Early Blight Introgressed in Potato.

Early blight is a disease of potato that is caused by Alternaria species, notably A. solani. The disease is usually controlled with fungicides. However, A. solani is developing resistance against fungicides, and potato cultivars with genetic resistance to early blight are currently not available. Here, we identify two wild potato species, which are both crossable with cultivated potato (Solanum tuberosum), that show promising resistance against early blight disease. The cross between resistant S. berthaultii and a susceptible diploid S. tuberosum gave rise to a population in which resistance was inherited quantitatively. S. commersonii subsp. malmeanum was also crossed with diploid S. tuberosum, despite a differing endosperm balance number. This cross resulted in triploid progeny in which resistance was inherited dominantly. This is somewhat surprising, as resistance against necrotrophic plant pathogens is usually a quantitative trait or inherited recessively according to the inverse-gene-for-gene model. Hybrids with high levels of resistance to early blight are present among progeny from S. berthaultii as well as S. commersonii subsp. malmeanum, which is an important step towards the development of a cultivar with natural resistance to early blight.

Quantifying the Contribution to Virulence of Phytophthora infestans Effectors in Potato.

Late blight in potato, caused by the oomycete Phytophthora infestans, is a devastating disease that significantly impacts potato production. For a proper understanding of disease development, it is important to understand the interaction between plant and pathogen at a molecular level. Like other pathogens, P. infestans secretes effector molecules, which can be recognized by receptors in the plant and trigger immunity. In addition, effectors from P. infestans have been identified to enhance disease development. Here, we describe an assay to investigate the role of effectors in virulence of P. infestans on potato. We combine agroinfiltration to transiently express effectors in potato with detached leaf assays to monitor disease development. This protocol makes it possible to conveniently quantify the effect of individual effectors on virulence of P. infestans. The identification of effectors with an important role in late blight development can help to design better strategies to control the disease.

Identification of Solanum Immune Receptors by Bulked Segregant RNA-Seq and High-Throughput Recombinant Screening.

The identification, understanding, and deployment of immune receptors are crucial to achieve high-level and durable resistance for crops against pathogens. In potato, many R genes have been identified using map-based cloning strategies. However, this is a challenging and laborious task that involves the development of a high number of molecular markers for the initial mapping, and the screening of thousands of plants for fine mapping. Bulked segregant RNA-Seq (BSR-Seq) has proven to be an efficient technique for the mapping of resistance genes. The RNA from two bulks of plants with contrasting phenotypes is sequenced and analyzed to identify single-nucleotide polymorphism (SNPs) markers linked to the target gene. Subsequently, the SNP markers that are identified can be used to delimit the mapping interval. Additionally, we designed an in vitro recombinant screening strategy that is advantageous for analyzing a large number of plants, in terms of time, space, and cost. Tips and detailed protocols, including BSR-Seq, bioinformatic analysis, and recombinant screening, are provided in this chapter.

RLP/K enrichment sequencing; a novel method to identify receptor-like protein (RLP) and receptor-like kinase (RLK) genes.

The identification of immune receptors in crop plants is time-consuming but important for disease control. Previously, resistance gene enrichment sequencing (RenSeq) was developed to accelerate mapping of nucleotide-binding domain and leucine-rich repeat containing (NLR) genes. However, resistances mediated by pattern recognition receptors (PRRs) remain less utilized. Here, our pipeline shows accelerated mapping of PRRs. Effectoromics leads to precise identification of plants with target PRRs, and subsequent RLP/K enrichment sequencing (RLP/KSeq) leads to detection of informative single nucleotide polymorphisms that are linked to the trait. Using Phytophthora infestans as a model, we identified Solanum microdontum plants that recognize the apoplastic effectors INF1 or SCR74. RLP/KSeq in a segregating Solanum population confirmed the localization of the INF1 receptor on chromosome 12, and led to the rapid mapping of the response to SCR74 to chromosome 9. By using markers obtained from RLP/KSeq in conjunction with additional markers, we fine-mapped the SCR74 receptor to a 43-kbp G-LecRK locus. Our findings show that RLP/KSeq enables rapid mapping of PRRs and is especially beneficial for crop plants with large and complex genomes. This work will enable the elucidation and characterization of the nonNLR plant immune receptors and ultimately facilitate informed resistance breeding.

Gapless Genome Assembly of the Potato and Tomato Early Blight Pathogen Alternaria solani.

The Alternaria genus consists of saprophytic fungi as well as plant-pathogenic species that have significant economic impact. To date, the genomes of multiple Alternaria species have been sequenced. These studies have yielded valuable data for molecular studies on Alternaria fungi. However, most of the current Alternaria genome assemblies are highly fragmented, thereby hampering the identification of genes that are involved in causing disease. Here, we report a gapless genome assembly of A. solani, the causal agent of early blight in tomato and potato. The genome assembly is a significant step toward a better understanding of pathogenicity of A. solani.

Discovering Novel Alternaria solani Succinate Dehydrogenase Inhibitors by in Silico Modeling and Virtual Screening Strategies to Combat Early Blight.

Alternaria blight is an important foliage disease caused by Alternaria solani. The enzyme Succinate dehydrogenase (SDH) is a potential drug target because of its role in tricarboxylic acid cycle. Hence targeting Alternaria solani SDH enzyme could be efficient tool to design novel fungicides against A. solani. We employed computational methodologies to design new SDH inhibitors using homology modeling; pharmacophore modeling and structure based virtual screening. The three dimensional SDH model showed good stereo-chemical and structural properties. Based on virtual screening results twelve commercially available compounds were purchased and tested in vitro and in vivo. The compounds were found to inhibit mycelial growth of A. solani. Moreover in vitro trials showed that inhibitory effects were enhanced with increase in concentrations. Similarly increased disease control was observed in pre-treated potato tubers. Hence the applied in silico strategy led us to identify novel fungicides.

Effectoromics-Based Identification of Cell Surface Receptors in Potato.

In modern resistance breeding, effectors have emerged as tools for accelerating and improving the identification of immune receptors. Effector-assisted breeding was pioneered for identifying resistance genes (R genes) against Phytophthora infestans in potato (Solanum tuberosum). Here we show that effectoromics approaches are also well suitable for identifying pathogen recognition receptors (PRRs) that recognize apoplastic effectors. To detect genotypes that recognize apoplastic proteins of P. infestans, routine agroinfiltration and potato virus X (PVX) agroinfection methods can be applied. In addition, protein infiltrations are feasible for assessing responses to apoplastic effectors and aid in confirming results obtained from the aforementioned methods. Protocols for the effectoromics pipeline are provided, starting from phenotyping for effector responses, up to genotyping and PRR gene identification.

Evidence for regulation of columnar habit in apple by a putative 2OG-Fe(II) oxygenase.

Understanding the genetic mechanisms controlling columnar-type growth in the apple mutant 'Wijcik' will provide insights on how tree architecture and growth are regulated in fruit trees. In apple, columnar-type growth is controlled by a single major gene at the Columnar (Co) locus. By comparing the genomic sequence of the Co region of 'Wijcik' with its wild-type 'McIntosh', a novel non-coding DNA element of 1956 bp specific to Pyreae was found to be inserted in an intergenic region of 'Wijcik'. Expression analysis of selected genes located in the vicinity of the insertion revealed the upregulation of the MdCo31 gene encoding a putative 2OG-Fe(II) oxygenase in axillary buds of 'Wijcik'. Constitutive expression of MdCo31 in Arabidopsis thaliana resulted in compact plants with shortened floral internodes, a phenotype reminiscent of the one observed in columnar apple trees. We conclude that MdCo31 is a strong candidate gene for the control of columnar growth in 'Wijcik'.

Post-translational regulation and trafficking of the granulin-containing protease RD21 of Arabidopsis thaliana.

RD21-like proteases are ubiquitous, plant-specific papain-like proteases typified by carrying a C-terminal granulin domain. RD21-like proteases are involved in immunity and associated with senescence and various types of biotic and abiotic stresses. Here, we interrogated Arabidopsis RD21 regulation and trafficking by site-directed mutagenesis, agroinfiltration, western blotting, protease activity profiling and protein degradation. Using an introduced N-glycan sensor, deglycosylation experiments and glyco-engineered N. benthamiana plants, we show that RD21 passes through the Golgi where it becomes fucosylated. Our studies demonstrate that RD21 is regulated at three post-translational levels. Prodomain removal is not blocked in the catalytic Cys mutant, indicating that RD21 is activated by a proteolytic cascade. However, RD21 activation in Arabidopsis does not require vacuolar processing enzymes (VPEs) or aleurain-like protease AALP. In contrast, granulin domain removal requires the catalytic Cys and His residues and is therefore autocatalytic. Furthermore, SDS can (re-)activate latent RD21 in Arabidopsis leaf extracts, indicating the existence of a third layer of post-translational regulation, possibly mediated by endogenous inhibitors. RD21 causes a dominant protease activity in Arabidopsis leaf extracts, responsible for SDS-induced proteome degradation.

Long-term effectiveness and costs of a brief self-management intervention in women with pregnancy-related low back pain after delivery.

BACKGROUND: Pregnancy-related low back pain is considered an important health problem and potentially leads to long-lasting pain and disability. Investigators draw particular attention to biomedical factors but there is growing evidence that psychosocial and social factors might be important. It prompted us to start a large cohort study (n = 7526) during pregnancy until one year after delivery and a nested randomized controlled intervention study in the Netherlands. METHODS: A randomized controlled trial (n = 126) nested within a cohort study, of brief self-management techniques versus usual care for treatment of women with persisting non-specific pregnancy-related low back pain three weeks after delivery. Women in the intervention group were referred to a participating physiotherapist. Women in the usual care group were free to choose physiotherapy, guidance by a general practitioner or no treatment. Follow up took place at 3 months, 6 months and one year after delivery.Outcomes included change in limitations in activities (RDQ), pain (VAS), severity of main complaints (MC), global feeling of recovery (GPE), impact on participation and autonomy (IPA), pain-related fear (TSK), SF-36, EuroQol and a cost diary. For the outcome measures, series of mixed models were considered. For the outcome variable global perceived effect (GPE) a logistic regression analysis is performed. RESULTS: Intention-to-treat outcomes showed a statistical significant better estimated regression coefficient RDQ -1.6 {-2.9;-0.5} associated with treatment, as well as better IPA subscale autonomy in self-care -1.0 {-1.9;-0.03} and TSK -2.4 {-3.8;-1.1} but were not clinical relevant over time. Average total costs in the intervention group were much lower than in usual care, primarily due to differences in utilization of sick leave but not statistically significant. CONCLUSION: Brief self-management techniques applied in the first 3 months after delivery may be a more viable first-line approach but further research is needed to draw inference on costs and to determine whether no care is a better option in the long term. TRIAL REGISTRATION: [ISRCTN08477490].

Low-frequency stimulation induces stable transitions in stereotypical activity in cortical networks.

Reverberating spontaneous synchronized brain activity is believed to play an important role in neural information processing. Whether and how external stimuli can influence this spontaneous activity is poorly understood. Because periodic synchronized network activity is also prominent in in vitro neuronal cultures, we used cortical cultures grown on multielectrode arrays to examine how spontaneous activity is affected by external stimuli. Spontaneous network activity before and after low-frequency electrical stimulation was quantified in several ways. Our results show that the initially stable pattern of stereotypical spontaneous activity was transformed into another activity pattern that remained stable for at least 1 h. The transformations consisted of changes in single site and culture-wide network activity as well as in the spatiotemporal dynamics of network bursting. We show for the first time that low-frequency electrical stimulation can induce long-lasting alterations in spontaneous activity of cortical neuronal networks. We discuss whether the observed transformations in network activity could represent a switch in attractor state.

Reproducibility and validity of the Dutch Life Habits Questionnaire (LIFE-H 3.0) in older adults.

OBJECTIVE: To examine the reproducibility, the discriminant and convergent validity and feasibility of the Dutch translation of the self-administered Life Habits Questionnaire (LIFE-H). DESIGN: Three cross-sectional community-based studies on clinimetric properties of a measurement instrument. SUBJECTS: Older adults (n=85) with functional limitations due to various chronic illnesses and healthy older adults (n=40). MEASUREMENT PROTOCOL: Participants of the reproducibility study (n=35) filled out the LIFE-H twice in a two-week time period. In the discriminant validity study (n=120), LIFE-H scores of healthy and ill subjects were compared. In the convergent validity study (n=63), correlations were examined between LIFE-H, the Impact on Participation and Autonomy questionnaire and the London Handicap Scale. RESULTS: The test-retest reliability showed a satisfactory intraclass correlation coefficient for the total overall score (0.80) but not for the categories scores. The discriminant validity study showed significant differences between the healthy and ill subjects for the 10 separate categories (P<0.01) and the total score (P<0.001). The correlations between the LIFE-H categories and total scores and the Impact on Participation and Autonomy Questionnaire (0.80-0.82) and London Handicap Scale (0.89-0.92) were strong. Feasibility testing showed that the subjects experienced difficulties due to the long and fairly complex instructions and structure of the LIFE-H. CONCLUSIONS: The clinimetric properties of the LIFE-H were moderate to good. The validity of LIFE-H was as good as the validity of the Impact on Autonomy and Participation and the London Handicap Scale, but the latter questionnaires were shorter and much easier to administer.

Effectiveness of a tailor-made intervention for pregnancy-related pelvic girdle and/or low back pain after delivery: short-term results of a randomized clinical trial [ISRCTN08477490].

BACKGROUND: For the moment, scientific evaluation of programs on treatment of pregnancy-related pelvic girdle and/or low back pain after delivery is hardly available with only one study with a positive result, suggesting uncertainty about the optimal approach. Investigators draw particular attention to biomedical factors but there is growing evidence that biopsychosocial factors appear to be even more important as a basis of an intervention program. METHODS: We studied the effectiveness of a tailor-made program with respect to biopsychosocial factors (intervention group) in women with pregnancy-related pelvic girdle and/ or low back pain versus usual care based on a pain contingent basis (control group) shortly after delivery in a randomized controlled trial. Women with severe complaints shortly after delivery were selected from a longitudinal prospective cohort study (n = 7526), aimed at pregnancy-related pelvic girdle and/or low back pain in the Netherlands. A concealed block randomization was performed after collecting baseline data. Researchers were blinded to treatment assignment. Outcomes were evaluated within the domains of the biopsychosocial approach. Primary outcome concerned limitations in activities (RDQ). Follow-up measurements were performed 12 weeks after delivery. RESULTS: Since May 2001 until July 2003, 869 women out of the cohort made a request for treatment by a physiotherapist, 10 days after delivery. Because of a quick recovery in two weeks time, we included only 126 women three weeks after delivery. There was a statistically significant and clinically relevant difference in improvement on the primary outcome (RDQ) between the two groups in favor of the experimental intervention. CONCLUSION: The results favored the hypotheses. Women's worries about their condition were major targets in the experimental intervention. The prognosis after delivery, especially in de first weeks, turned out to be favorable.

Health care providers' orientations towards common low back pain predict perceived harmfulness of physical activities and recommendations regarding return to normal activity.

The Pain Attitudes and Beliefs Scale for Physiotherapists (PABS-PT) differentiates between a biomedical versus a biopsychosocial treatment orientation with regard to common low back pain. This study re-examined the factor structure and psychometric properties of the PABS-PT, along with the relationship between PABS-PT scores and the perceived harmfulness of physical activities and treatment recommendations for common low back pain. Two hundred and ninety-seven paramedical therapists completed the PABS-PT and questionnaires measuring related concepts, rated the perceived harmfulness of 41 daily physical activities depicted in photographs and gave recommendations for return to normal activity for three patients with low back pain. Analysis revealed two factors labelled 'biomedical' and 'biopsychosocial treatment orientation'. Furthermore, scores on both factors of the PABS-PT were related to measures of related concepts (statistically significant Pearson correlation coefficients between 0.30 and 0.65) such as the HC-PAIRS and a therapist version of the TSK. Regression analyses revealed that both factors were consistent predictors of judgements of the harmfulness of physical activities (PHODA) and of recommendations for return to work and normal activity.

Dynamics and plasticity in developing neuronal networks in vitro.

When dissociated cortical tissue is brought into culture, neurons readily grow out by forming axonal and dendritic arborizations and synaptic connections. These developing neuronal networks in vitro display spontaneous firing activity from about the end of the first week in vitro. When cultured on multielectrode arrays firing activity can be recorded from many neurons simultaneously over long periods of time. These experimental approaches provide valuable data for studying firing dynamics in neuronal networks in relation to an ongoing development of neurons and synaptic connectivity in the network. This chapter summarizes recent findings on the characteristics and developmental changes in the spontaneous firing dynamics. These changes include long-lasting transient periods of increased firing at individual sites on a time scale of days to weeks, and an age-specific repetitive pattern of synchronous network firing (network bursts) on a time scale of seconds. Especially the spatio-temporal organization of firing within network bursts showed great stability over many hours. In addition, a progressive day-to-day evolution was observed, with an initial broadening of the burst firing rate profile during the 3rd week in vitro (WIV) and a pattern of abrupt onset and precise spike timing from the 5th WIV onwards. These developmental changes are discussed in the light of structural changes in the network and activity-dependent plasticity mechanisms. Preliminary findings are presented on the pattern of spike sequences within network burst, as well as the effect of external stimulation on the spatio-temporal organization within network bursts.

Compensatory physiological responses to chronic blockade of amino acid receptors during early development in spontaneously active organotypic cerebral cortex explants cultured in vitro.

Paired organotypic explants from rat occipital cortex were cultured for up to three weeks in the presence of selective blockers of amino acid receptor blockers, during which period spontaneous action potential generation was monitored electrophysiologically. In contrast to isolated explants (Corner, M.A., van Pelt, J., Wolters, P.S., Baker, R.E.and Nuytinck, R.H. (2002) Physiological e.ects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks--an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny. Neurosci. Biobehav. Rev., 26: 127-185), which upregulated their initially depressed spontaneous bursting activity only under conditions of N-methyl D-aspartate (NMDA) receptor blockade, cross-innervated co-cultures showed a large degree of functional recovery even when combined NMDA and AMPA receptor blockade was carried out. This compensatory activity could be eliminated by acute addition of a selective kainate receptor blocker to the medium. When kainate along with AMPA and NMDA receptor mediated activity was chronically suppressed, however, considerable functional recovery--in the form of recurrent burst discharges--took place gradually over a period of three weeks in vitro. These spontaneous bursts disappeared rapidly upon treatment with the muscarinic receptor blocker, atropine, but continuous low-level firing emerged at the same time. Similar "tonic" background activity was induced in control cultures as well, but without any noticeable reduction in burst discharges. Co-cultured neocortex explants, in which cyto-morphological maturation proceeds to a far greater degree than in isolated explants (Baker, R.E.and van Pelt, J. (1997) Co-cultured but not isolated cortical explants display normal dendritic development: a longterm quantitative study. Dev. Brain Res., 98: 21-27) are evidently capable of an astonishing degree of functional compensation for loss of excitatory synaptic drive during development. It could be shown, furthermore, that such homeostatic responses are not mediated largely by a weakening of inhibitory mechanisms in the absence of spontaneous firing. Chronic inhibitory synaptic blockade, on the other hand, led to intensified bursting activity which gradually normalized over a 3-week culture period. The cellular basis for this reversal of the disinhibited state, as well as for the residual neuronal firing even after cholinergic mechanisms have been largely eliminated, is at present unknown. The degree to which immature cortical networks attempt to compensate for altered levels of physiological activity, as documented in the present report, is another indication of how important such activity can be for normal development (see Corner, M.A., van Pelt, J., Wolters, P.S., Baker, R.E. and Nuytinck, R.H. (2002) Physiological e.ects of sustained blockade of excitatory synaptic transmission on spontaneously active developing neuronal networks-an inquiry into the reciprocal linkage between intrinsic biorhythms and neuroplasticity in early ontogeny. Neurosci. Biobehav. Rev., 26: 127-185).. At the same time, the large variations in overall firing levels and "macro-scale" temporal patterns from culture to culture within a given series, despite all attempts at identical preparation of the explants, can only mean that the "set-points" for such regulation are themselves subject to unknown ontogenetic factors which, apparently, are nonuniformly distributed even within a restricted region of the neocortex. On the other hand, it was striking to note that, regardless of age or treatment, an unexpected degree of consistency in temporal patterning existed at "mini-" and "micro-" time-scales (viz., EEG delta and beta frequency ranges, respectively) even when network bursting tendencies became greatly reduced in favor of tonic firing.