Type 2 diabetes mellitus facilitates status epilepticus in adult rats: Seizure severity, neurodegeneration, and oxidative stress.

OBJECTIVE: The goal of this research was to evaluate the effect of DM type 2 (DM2) on SE severity, neurodegeneration, and brain oxidative stress (OS) secondary to seizures. METHODS: DM2 was induced in postnatal day (P) 3 male rat pups by injecting streptozocin (STZ) 100 mg/kg; control rats were injected with citrate buffer as vehicle. At P90, SE was induced by the lithium-pilocarpine administration and seizure latency, frequency, and severity were evaluated. Neurodegeneration was assessed 24 h after SE by Fluoro-Jade B (F-JB) staining, whereas OS was estimated by measuring lipid peroxidation and reactive oxygen species (ROS). RESULTS: DM2 rats showed an increase in latency to the first generalized seizure and SE onset, had a higher number and a longer duration of seizures, and displayed a larger neurodegeneration in the hippocampus (CA3, CA1, dentate gyrus, and hilus), the piriform cortex, the dorsomedial nucleus of the thalamus and the cortical amygdala. Our results also show that only SE, neither DM2 nor the combination of DM2 with SE, caused the increase in ROS and brain lipid peroxidation. SIGNIFICANCE: DM2 causes higher seizure severity and neurodegeneration but did not exacerbate SE-induced OS under these conditions. PLAIN LANGUAGE SUMMARY: Our research performed in animal models suggests that type 2 diabetes mellitus (DM2) may be a risk factor for causing higher seizure severity and seizure-induced neuron cell death. However, even when long-term seizures promote an imbalance between brain pro-oxidants and antioxidants, DM2 does not exacerbate that disproportion.

Synchronization of inspiratory burst onset along the ventral respiratory column in the neonate mouse is mediated by electrotonic coupling.

Breathing is a singularly robust behavior, yet this motor pattern is continuously modulated at slow and fast timescales to maintain blood-gas homeostasis, while intercalating orofacial behaviors. This functional multiplexing goes beyond the rhythmogenic function that is typically ascribed to medullary respiration-modulated networks and may explain lack of progress in identifying the mechanism and constituents of the respiratory rhythm generator. By recording optically along the ventral respiratory column in medulla, we found convergent evidence that rhythmogenic function is distributed over a dispersed and heterogeneous network that is synchronized by electrotonic coupling across a neuronal syncytium. First, high-speed recordings revealed that inspiratory onset occurred synchronously along the column and did not emanate from a rhythmogenic core. Second, following synaptic isolation, synchronized stationary rhythmic activity was detected along the column. This activity was attenuated following gap junction blockade and was silenced by tetrodotoxin. The layering of syncytial and synaptic coupling complicates identification of rhythmogenic mechanism, while enabling functional multiplexing.

Modulation of inspiratory burst duration and frequency by bombesin in vitro.

Mammalian respiratory rhythm-generating circuits in the brainstem are subject to neuromodulation by multiple peptidergic afferent inputs controlling circuit behavior and outputs. Although functionally important, actions of neuropeptide modulators have not been fully characterized. We analyzed at cellular and circuit levels two inspiratory patterns intrinsically generated by the preBötzinger complex (preBötC) and their modulation by the neuropeptides bombesin and substance P (SP) in neonatal rat medullary slices in vitro. We found that, in recordings of hypoglossal nerve and preBötC neuron inspiratory activity, some inspiratory bursts occurring spontaneously under basal conditions have a biphasic shape with longer duration than normal inspiratory bursts and occur at a lower frequency. This biphasic burst pattern has been proposed to represent inspiratory activity underling periodic sighs. Bath-applied bombesin or SP decreased the period and increased the duration of both normal inspiratory and biphasic bursts and their underlying synaptic drives. The ratio of the biphasic long-duration burst period to the normal inspiratory burst period and the ratio of their burst durations remained the same before and after peptidergic modulation. Bombesin increased the frequency of the inspiratory rhythm in a Ca2+-independent manner and the frequency of long-duration bursts in a Ca2+-dependent manner. This finding suggests that period and burst duration coupling are due to intrinsic mechanisms controlling simultaneously timing and burst termination within the inspiratory rhythm-generating network. We propose a model in which signaling cascades activated by bombesin and SP modulate mechanisms controlling inspiratory burst frequency and duration to coordinate preBötC circuit behavioral outputs.

Transgenic rodents as dynamic models for the study of respiratory rhythm generation and modulation: a scoping review and a bibliometric analysis.

The pre-Bötzinger complex, situated in the ventrolateral medulla, serves as the central generator for the inspiratory phase of the respiratory rhythm. Evidence strongly supports its pivotal role in generating, and, in conjunction with the post-inspiratory complex and the lateral parafacial nucleus, in shaping the respiratory rhythm. While there remains an ongoing debate concerning the mechanisms underlying these nuclei's ability to generate and modulate breathing, transgenic rodent models have significantly contributed to our understanding of these processes. However, there is a significant knowledge gap regarding the spectrum of transgenic rodent lines developed for studying respiratory rhythm, and the methodologies employed in these models. In this study, we conducted a scoping review to identify commonly used transgenic rodent lines and techniques for studying respiratory rhythm generation and modulation. Following PRISMA guidelines, we identified relevant papers in PubMed and EBSCO on 29 March 2023, and transgenic lines in Mouse Genome Informatics and the International Mouse Phenotyping Consortium. With strict inclusion and exclusion criteria, we identified 80 publications spanning 1997-2022 using 107 rodent lines. Our findings revealed 30 lines focusing on rhythm generation, 61 on modulation, and 16 on both. The primary in vivo method was whole-body plethysmography. The main in vitro method was hypoglossal/phrenic nerve recordings using the en bloc preparation. Additionally, we identified 119 transgenic lines with the potential for investigating the intricate mechanisms underlying respiratory rhythm. Through this review, we provide insights needed to design more effective experiments with transgenic animals to unravel the mechanisms governing respiratory rhythm. The identified transgenic rodent lines and methodological approaches compile current knowledge and guide future research towards filling knowledge gaps in respiratory rhythm generation and modulation.

An open-source low-cost wireless sensor system for acquisition of human movement data.

Several fields of research such as medicine, robotics, sports, informatics, etc., require the analysis of human movement. Traditional systems for acquisition and analysis of human movement data are based on video cameras or active sensors. However, those systems are limited to high-resource settings. Wearable devices allow monitoring subjects outside typical clinical or research environments. Here, we present an open source low-cost wireless sensor system for acquisition of human movement data. Our system consists of two main parts: a server that stores data and, one or more wearable sensor modules that collect movement data through Inertial Measurement Units (IMUs) and transmit them wirelessly to the server. As a proof of concept, we measured human gait activity. Our results show that our system with IMUs can acquire quantifiable movement data. Characteristics such as open source code and its low-cost, make our system a viable alternative for clinical or research.

Analixity: An open source, low-cost analysis system for the elevated plus maze test, based on computer vision techniques.

Manual analysis of behavioral tests in rodents involves inspection of video recordings by a researcher that assesses rodent movements to quantify parameters related with a behavior of interest. The assessment of the researcher during the quantification of such parameters can introduce variability among experimental conditions or among sessions of analysis. Here, we introduce Analixity, a video processing software for the elevated plus maze test (EPM), in which quantification of behavioral parameters is automatic, reducing the time spent in analysis and solving the variability problem. Analixity is an adaptable multiplatform open-source system. Analixity generates an Excel file with the quantified behavioral variables, such as time spent in open and closed arms and in the center zone, number of entries to each zone and total distance traveled during the test. For validation, we compared results obtained by Analixity with results obtained by manual analysis. We did not find statistically significant differences. In addition, we compared the results obtained by Analixity with results obtained by the commercial software ANY-maze. We did not find statistically significant differences in the quantification of parameters such as time spent in open arms, time spent in closed arms, time spent in center zone, number of closed arms, open arms entries, and anxiety index. We concluded that Analixity is an open-source software as reliable and effective as a commercial software.

Anxiolytic effect of chronic intake of supplemental magnesium chloride in rat.

Evidence suggest that magnesium dietary supplementation has several health benefits including lowering blood pressure, reducing insulin resistance, and improving symptoms of depression, anxiety, and migraine. Here, we aimed to study the effect of chronic magnesium supplementation on anxiety-like behavior in rats by supplementing with magnesium their drinking water for 30 days. Anxiety-like behavior was induced by subcutaneous injection of veratrin 30 min before performing elevated plus maze and open field tests to measure anxiety levels and locomotion, respectively. We quantify the concentration of magnesium in plasma and cerebrospinal fluid. We used diazepam to compare the efficacy of magnesium supplementation as an anxiolytic agent. Our results show that rats supplemented with magnesium had a statistically significant decrease in anxiety levels with not effects on locomotion and a statistically significant increase in concentration of magnesium in plasma and cerebrospinal fluid. However, the anxiolytic effect of magnesium supplementation washes-out in 12 days. We discuss the advantages of using supplemental magnesium as anxiolytic.

Brain and plasma amino acid concentration in infant rats prenatally exposed to valproic acid.

Autism spectrum disorder is associated with alterations in GABAergic and glutamatergic neurotransmission. Here, we aimed to determine the concentration of GABA, glutamate, glutamine, aspartate, taurine, and glycine in brain tissue and plasma of rats prenatally exposed to valproic acid (VPA), a well-characterized experimental model of autism. Pregnant rats were injected with VPA (600mg/Kg) during the twelfth-embryonic-day. Control rats were injected with saline. On the fourteen-postnatal-day, rats from both groups (males and females) were anesthetized, euthanized by decapitation and their brain dissected out. The frontal cortex, hippocampus, amygdala, brain stem and cerebellum were dissected and homogenized. Homogenates were centrifuged and supernatants were used to quantify amino acid concentrations by HPLC coupled with fluorometric detection. Blood samples were obtained by a cardiac puncture; plasma was separated and deproteinized to quantify amino acid concentration by HPLC. We found that, in VPA rats, glutamate and glutamine concentrations were increased in hippocampus and glycine concentration was increased in cortex. We did not find changes in other regions or in plasma amino acid concentration in the VPA group with respect to control group. Our results suggest that VPA exposure in utero may impair inhibitory and excitatory amino acid transmission in the infant brain.

Longitudinal Analysis of the Relation Between Clinical Impairment and Gray Matter Degeneration in Spinocerebellar Ataxia Type 7 Patients.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by progressive ataxia and retinal degeneration. Previous cross-sectional studies show a significant decrease in the gray matter of the cerebral cortex, cerebellum, and brainstem. However, there are no longitudinal studies in SCA7 analyzing whole-brain degeneration and its relation to clinical decline. To perform a 2-year longitudinal characterization of the whole-brain degeneration and clinical decline in SCA7, twenty patients underwent MRI and clinical evaluations at baseline. Fourteen completed the 2-year follow-up study. A healthy-matched control group was also included. Imaging analyses included volumetric and cortical thickness evaluation. We measured the cognitive deterioration in SCA7 patients using MoCA test and the motor deterioration using the SARA score. We found statistically significant differences in the follow-up compared to baseline. Imaging analyses showed that SCA7 patients had severe cerebellar and pontine degeneration compared with the control group. Longitudinal follow-up imaging analyses of SCA7 patients showed the largest atrophy in the medial temporal lobe without signs of a progression of cerebellar and pontine atrophy. Effect size analyses showed that MRI longitudinal analysis has the largest effect size followed by the SARA scale and MoCA test. Here, we report that it is possible to detect significant brain atrophy and motor and cognitive clinical decline in a 2-year follow-up study of SCA7 patients. Our results support the hypothesis that longitudinal analysis of structural MRI and MOCA tests are plausible clinical markers to study the natural history of the disease and to design treatment trials in ecologically valid contexts.

Sleep Recovery Restored Neuroglobin Immunoreactivity in Rat LDTg-PPTg Nuclei.

Neuroglobin (Ngb) is a protein member of the globin family, expressed mainly in the central and peripheral nervous system. It is involved in the transport of oxygen in response to hypoxic/ischemic and oxidative stress-related insults. We recently showed that sleep deprivation reduces the number of Ngb-positive cells in brain areas related to sleep. However, it is poorly understood whether Ngb expression correlates with sleep occurrence. Here, we aimed to study if sleep recovery produced by 24 h of sleep deprivation restores the number of Ngb-positive cells in the pedunculopontine tegmentum (PPTg) and laterodorsal tegmentum (LDTg), brain areas related to sleep-wake regulation. Male Wistar rats were sleep-deprived for 24 h using the gentle handling method. After sleep deprivation, rats were allowed a sleep recovery for three or six hours. After sleep recovery, rats were euthanized, and their brains processed for Ngb immunohistochemistry. We found that a 3 h sleep recovery is enough to restore the number of Ngb-positive cells in all the analyzed areas. A similar result was observed after a 6 h sleep recovery. These results suggest that Ngb expression is sleep dependent. We suggest that Ngb expression is involved in preventing cell damage due to prolonged wakefulness.

Characterization and Classification of Electrophysiological Signals Represented as Visibility Graphs Using the Maxclique Graph.

Detection, characterization and classification of patterns within time series from electrophysiological signals have been a challenge for neuroscientists due to their complexity and variability. Here, we aimed to use graph theory to characterize and classify waveforms within biological signals using maxcliques as a feature for a deep learning method. We implemented a compact and easy to visualize algorithm and interface in Python. This software uses time series as input. We applied the maxclique graph operator in order to obtain further graph parameters. We extracted features of the time series by processing all graph parameters through K-means, one of the simplest unsupervised machine learning algorithms. As proof of principle, we analyzed integrated electrical activity of XII nerve to identify waveforms. Our results show that the use of maxcliques allows identification of two distinct types of waveforms that match expert classification. We propose that our method can be a useful tool to characterize and classify other electrophysiological signals in a short time and objectively. Reducing the classification time improves efficiency for further analysis in order to compare between treatments or conditions, e.g., pharmacological trials, injuries, or neurodegenerative diseases.

Status Epilepticus Increases Cell Proliferation and Neurogenesis in the Developing Rat Cerebellum.

Status epilepticus (SE) promotes neuronal proliferation and differentiation in the adult and developing rodent hippocampus. However, the effect of SE on other neurogenic brain regions such as the cerebellum has been less explored. To determine whether SE induced by pentylentetrazole (PTZ-SE) and lithium-pilocarpine (Li-Pilo-SE) increases cell proliferation and neurogenesis in the developing rat cerebellum. SE was induced in 14-day-old (P14) Wistar rat pups (both sexes). One hour after SE and the following day rats were injected intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU, 50 mg/kg). Seven days after SE, immunohistochemistry was performed to detect BrdU-positive (BrdU+) cells or BrdU/NeuN+ cells in the cerebellar vermis. SE induced by PTZ or Li-Pilo statistically significant increased the number of cerebellar BrdU+ cells when compared with the control group (58% and 40%, respectively); maximal cell proliferation occurred in lobules II, III, VIb, VIc, VIII, IXa, and IXb of PTZ-SE group and II, V, VIc, VII, and X of Li-Pilo-SE group. An increased number of BrdU/NeuN+ cells was detected in lobules V (17 ± 1.9), VIc (25.8 ± 2.7), and VII (26.2 ± 3.4) after Li-Pilo-SE compared to their control group (9.8 ± 1.7, 12.8 ± 2.8, and 11 ± 1.7, respectively), while the number of BrdU/NeuN+ cells remained the same after PTZ-induced SE or control conditions. SE induced in the developing rat by different experimental models increases cell proliferation in the granular layer of the cerebellar vermis, but only SE of limbic seizures increases neurogenesis in specific cerebellar lobes.

Differential Expression of Ion Channels in Adult and Neonatal Rat Ventral Respiratory Column.

In mammals, the neural control of breathing is attributed to circuits distributed along the ventral respiratory column (VRC) in the ventrolateral medulla. The VRC contains the kernel for generation of the inspiratory phase of respiratory rhythm and nuclei involved in central chemoreception. During development, the respiratory rhythm, as well as central chemosensitivity, adjusts to meet the changing physiological requirements associated with increased body weight and size. Gene expression in VRC ontogeny is well characterized. However, little is known about gene expression in the VRC during postnatal development. Here, we sought to characterize the changes in gene expression that occur in the VRC of the adult rat (5-6 months of age) in comparison with the VRC of neonate rat (1-4 days old). We isolated total RNA from VRC tissue punches collected from thick transversal slices. We hybridized cDNA to a 5000-oligonucleotide rat microarray. We found that 218 genes (4.4%) of the 5000 genes in the microarray changed their expression in adult VRC with respect to that from neonate. To further analyze the modified expression of specific genes, we quantified the differential expression of 84 genes of neuronal ion channels using a quantitative RT-PCR array. This analysis confirmed the overexpression of 68 genes and the underexpression of 14 genes in the VRC from adult compared with that from neonate. Our findings may help to explain the functional changes in respiratory rhythm and chemosensitivity occurring throughout life.

Respiratory Rhythm Generation: The Whole Is Greater Than the Sum of the Parts.

Breathing is a continuous behavior essential for life in mammals and one of the few behaviors that can be studied in vivo in intact animals awake, anesthetized or decerebrated and in highly reduced in vitro and in situ preparations. The preBötzinger complex (preBötC) is a small nucleus in the brainstem that plays an essential role in normal breathing and is widely accepted as the site necessary and sufficient for generation of the inspiratory phase of the respiratory rhythm. Substantial advances in understanding the anatomical and cellular basis of respiratory rhythmogenesis have arisen from in vitro and in vivo studies in the past 25 years; however, the underlying cellular mechanisms remain unknown.

The Onset of the Fetal Respiratory Rhythm: An Emergent Property Triggered by Chemosensory Drive?

The mechanisms responsible for the onset of respiratory activity during fetal life are unknown. The onset of respiratory rhythm may be a consequence of the genetic program of each of the constituents of the respiratory network, so they start to interact and generate respiratory cycles when reaching a certain degree of maturation. Alternatively, generation of cycles might require the contribution of recently formed sensory inputs that will trigger oscillatory activity in the nascent respiratory neural network. If this hypothesis is true, then sensory input to the respiratory generator must be already formed and become functional before the onset of fetal respiration. In this review, we evaluate the timing of the onset of the respiratory rhythm in comparison to the appearance of receptors, neurotransmitter machinery, and afferent projections provided by two central chemoreceptive nuclei, the raphe and locus coeruleus nuclei.

Pentylenetetrazole-induced seizures in developing rats prenatally exposed to valproic acid.

BACKGROUND: Epidemiological evidence indicates epilepsy is more common in patients with autism spectrum disorders (ASD) (20-25%) than in the general population. The aim of this project was to analyze seizure susceptibility in developing rats prenatally exposed to valproic acid (VPA) as autism model. METHODS: Pregnant females were injected with VPA during the twelfth embryonic day. Seizures were induced in fourteen-days-old rat pups using two models of convulsions: pentylenetetrazole (PTZ) and lithium-pilocarpine (Li-Pilo). RESULTS: Two subgroups with different PTZ-induced seizure susceptibility in rats exposed to VPA were found: a high susceptibility (VPA+) (28/42, seizure severity 5) and a low susceptibility (VPA-) (14/42, seizure severity 2). The VPA+ subgroup exhibited an increased duration of the generalized tonic-clonic seizure (GTCS; 45 ± 2.7 min), a higher number of rats showed several GTCS (14/28) and developed status epilepticus (SE) after PTZ injection (19/27) compared with control animals (36.6 ± 1.9 min; 10/39; 15/39, respectively). No differences in seizure severity, latency or duration of SE induced by Li-Pilo were detected between VPA and control animals. DISCUSSION: Prenatal VPA modifies the susceptibility to PTZ-induced seizures in developing rats, which may be linked to an alteration in the GABAergic transmission. These findings contribute to a better understanding of the comorbidity between autism and epilepsy.

Substitution of extracellular Ca2+ by Sr2+ prolongs inspiratory burst in pre-Bötzinger complex inspiratory neurons.

The pre-Bötzinger complex (preBötC) underlies inspiratory rhythm generation. As a result of network interactions, preBötC neurons burst synchronously to produce rhythmic premotor inspiratory activity. Each inspiratory burst consists of action potentials (APs) on top of a 10- to 20-mV synchronous depolarization lasting 0.3-0.8 s known as inspiratory drive potential. The mechanisms underlying the initiation and termination of the inspiratory burst are unclear, and the role of Ca(2+) is a matter of intense debate. To investigate the role of extracellular Ca(2+) in inspiratory burst initiation and termination, we substituted extracellular Ca(2+) with Sr(2+). We found for the first time an ionic manipulation that significantly interferes with burst termination. In a rhythmically active slice, we current-clamped preBötC neurons (Vm ≅ -60 mV) while recording integrated hypoglossal nerve (∫XIIn) activity as motor output. Substitution of extracellular Ca(2+) with either 1.5 or 2.5 mM Sr(2+) significantly prolonged the duration of inspiratory bursts from 653.4 ± 30.7 ms in control conditions to 981.6 ± 78.5 ms in 1.5 mM Sr(2+) and 2,048.2 ± 448.5 ms in 2.5 mM Sr(2+), with a concomitant increase in decay time and area. Substitution of extracellular Ca(2+) by Sr(2+) is a well-established method to desynchronize neurotransmitter release. Our findings suggest that the increase in inspiratory burst duration is determined by a presynaptic mechanism involving desynchronization of glutamate release within the network.

IL-1ß increases necrotic neuronal cell death in the developing rat hippocampus after status epilepticus by activating type I IL-1 receptor (IL-1RI).

Interleukin-1ß (IL-1ß) is associated with seizure-induced neuronal cell death in the adult brain. The contribution of IL-1ß to neuronal injury induced by status epilepticus (SE) in the immature brain remains unclear. In the present study, we investigated the effects of IL-1ß administration on hippocampal neuronal cell death associated with SE in the immature brain, and the role of the type I receptor of IL-1ß (IL-1RI). SE was induced with lithium-pilocarpine in 14-days-old (P14) rat pups. Six hours after SE onset, pups were i.c.v. injected in the right ventricle with IL-1ß (0, 0.3, 3, 30, or 300 ng), 30 ng of IL-1RI antagonist (IL-1Ra) alone, or 30 ng of IL-1Ra plus 3ng of IL-1ß. As control groups, pups without seizures were injected with 3 ng of IL-1ß or vehicle. Twenty-four hours after SE onset, neuronal cell death in the CA1 field of dorsal hippocampus was assessed by hematoxylin-eosin, Fluoro-Jade B and in vivo propidium iodide (PI) staining; expression of active caspase-3 (aCas-3) was also determined, using immunohistochemistry. The concentration-response curve of IL-1ß showed a bell-shape. Only pups injected with 3 ng of IL-1ß after SE showed a significant increase in the number of cells with eosinophilic cytoplasm and pyknotic nuclei, as well as F-JB positive cells with respect to the vehicle group. This effect was prevented when IL-1ß was injected with IL-1Ra. Injection of 3 ng of IL-1ß increased the number of PI-positive cells in CA1 area after SE. Injection of 3 ng of IL-1ß did not produce hippocampal cell death in rats without seizures. Active caspase-3 expression was not observed after treatments in hippocampus. The activation of the IL-1ß/IL-1RI system increases necrotic neuronal cell death caused by SE in rat pups.

Design and construction of a modular low-cost epifluorescence upright microscope for neuron visualized recording and fluorescence detection.

BACKGROUND: One of the limitations when establishing an electrophysiology setup, particularly in low resource settings, is the high cost of microscopes. The average cost for a microscope equipped with the optics for infrared (IR) contrast or microfluorometry is $40,000. We hypothesized that optical elements and features included in commercial microscopes are not necessary to IR video-visualize neurons or for microfluorometry. NEW METHOD: We present instructions for building a low-cost epifluorescence upright microscope suitable for visualized patch-clamp recording and fluorescence detection using mostly catalog-available parts. RESULTS: This microscope supports applications such as visualized whole-cell recording using IR oblique illumination (IR-OI), or more complex applications such as microfluorometry using a photodiode. In both IR-OI and fluorescence, actual resolution measured with 2-µm latex beads is close to theoretical resolution. The lack of movable parts to switch configurations ensures stability when doing intracellular recording. COMPARISON WITH EXISTING METHODS: The low cost is a significant advantage of this microscope compared to existent custom-built microscopes. The cost of the simplest configuration with IR-OI is ∼$2000, whereas the cost of the configuration with epifluorescence is ∼$5000. Since this design does not use pieces discarded from commercial microscopes, it is completely reproducible. CONCLUSIONS: We suggest that this microscope is a viable alternative for doing in vitro electrophysiology and microfluorometry in low-resource settings. Characteristics such as an open box design, easy assembly, and low-cost make this microscope a useful instrument for science education and teaching for topics such as optics, biology, neuroscience, and for scientific "hands-on" workshops.