Unraveling the assembloid: Real-time monitoring of dopaminergic neurites in an inter-organoid pathway connecting midbrain and striatal regions.

Modern in vitro technologies for preclinical research, including organ-on-a-chip, organoids- and assembloid-based systems, have rapidly emerged as pivotal tools for elucidating disease mechanisms and assessing the efficacy of putative therapeutics. In this context, advanced in vitro models of Parkinson's Disease (PD) offer the potential to accelerate drug discovery by enabling effective platforms that recapitulate both physiological and pathological attributes of the in vivo environment. Although these systems often aim at replicating the PD-associated loss of dopaminergic (DA) neurons, only a few have modelled the degradation of dopaminergic pathways as a way to mimic the disruption of downstream regulation mechanisms that define the characteristic motor symptoms of the disease. To this end, assembloids have been successfully employed to recapitulate neuronal pathways between distinct brain regions. However, the investigation and characterization of these connections through neural tracing and electrophysiological analysis remain a technically challenging and time-consuming process. Here, we present a novel bioengineered platform consisting of surface-grown midbrain and striatal organoids at opposite sides of a self-assembled DA pathway. In particular, dopaminergic neurons and striatal GABAergic neurons spontaneously form DA connections across a microelectrode array (MEA), specifically integrated for the real-time monitoring of electrophysiological development and stimuli response. Calcium imaging data showed spiking synchronicity of the two organoids forming the inter-organoid pathways (IOPs) demonstrating that they are functionally connected. MEA recordings confirm a more robust response to the DA neurotoxin 6-OHDA compared to midbrain organoids alone, thereby validating the potential of this technology to generate highly tractable, easily extractable real-time functional readouts to investigate the dysfunctional dopaminergic network of PD patients.

Biomaterials-based strategies for in vitro neural models.

In vitro models have been used as a complementary tool to animal studies in understanding the nervous system's physiological mechanisms and pathological disorders, while also serving as platforms to evaluate the safety and efficiency of therapeutic candidates. Following recent advances in materials science, micro- and nanofabrication techniques and cell culture systems, in vitro technologies have been rapidly gaining the potential to bridge the gap between animal and clinical studies by providing more sophisticated models that recapitulate key aspects of the structure, biochemistry, biomechanics, and functions of human tissues. This was made possible, in large part, by the development of biomaterials that provide cells with physicochemical features that closely mimic the cellular microenvironment of native tissues. Due to the well-known material-driven cellular response and the importance of mimicking the environment of the target tissue, the selection of optimal biomaterials represents an important early step in the design of biomimetic systems to investigate brain structures and functions. This review provides a comprehensive compendium of commonly used biomaterials as well as the different fabrication techniques employed for the design of neural tissue models. Furthermore, the authors discuss the main parameters that need to be considered to develop functional platforms not only for the study of brain physiological functions and pathological processes but also for drug discovery/development and the optimization of biomaterials for neural tissue engineering.

[11C]meta-hydroxyephedrine PET evaluation in experimental pulmonary arterial hypertension: Effects of carvedilol of right ventricular sympathetic function.

BACKGROUND: Little is known about the sequelae of chronic sympathetic nervous system (SNS) activation in patients with pulmonary arterial hypertension (PAH) and right heart failure (RHF). We aimed to, (1) validate the use of [11C]-meta-hydroxyephedrine (HED) for assessing right ventricular (RV) SNS integrity, and (2) determine the effects of ß-receptor blockade on ventricular function and myocardial SNS activity in a PAH rat model. METHODS: PAH was induced in male Sprague-Dawley rats (N = 36) using the Sugen+chronic hypoxia model. At week 5 post-injection, PAH rats were randomized to carvedilol (15 mg·kg-1·day-1 oral; N = 16) or vehicle (N = 16) for 4 weeks. Myocardial SNS function was assessed with HED positron emission tomography(PET). RESULTS: With increasing PAH disease severity, immunohistochemistry confirmed selective sympathetic denervation within the RV and sparing of parasympathetic nerves. These findings were confirmed on PET with a significant negative relationship between HED volume of distribution(DV) and right ventricular systolic pressure (RVSP) in the RV (r = -0.90, p = 0.0003). Carvedilol did not reduce hemodynamic severity compared to vehicle. RV ejection fraction (EF) was lower in both PAH groups compared to control (p < 0.05), and was not further reduced by carvedilol. Carvedilol improved SNS function in the LV with significant increases in the HED DV, and decreased tracer washout in the LV (p < 0.05) but not RV. CONCLUSIONS: PAH disease severity correlated with a reduction in HED DV in the RV. This was associated with selective sympathetic denervation. Late carvedilol treatment did not lead to recovery of RV function. These results support the role of HED imaging in assessing SNS innervation in a failing right ventricle.

A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling.

The typical immunohistochemistry technique used to reveal 5-bromo-2'-deoxyuridine (BrdU) incorporation requires denaturation of the DNA by heat and acid to permeabilize the cell nucleus. This treatment can damage tissue and reduce the antigenicity of several proteins, which then leads to weak immunostaining and/or false negatives. We show that an overnight post-fixation step following immunohistochemistry for antigens of interest protects immunostaining during the acid/heat denaturation treatment for subsequent BrdU staining. We used this technique to study the differentiation of recently divided oligodendrocyte progenitor cells in NG2CreER:EYFP reporter mice. We used a GFP anti-EYFP antibody to maximize visualization of the EYFP-containing oligodendrocyte progenitor cells, Olig1, and GST-pi to confirm the cell phenotype. Immunostaining for GFP, Olig1, and GST-pi is reduced by DNA denaturation. We found that incorporating a post-fixation step after double immunostaining for GFP/Olig1 and GFP/GST-pi prior to DNA denaturation prevented the fading and false negatives associated with this treatment. This simple addition to BrdU immunohistochemistry protocols extends the range of proteins that can be detected in combination with BrdU, along with the number of antibodies that can be used successfully in the study of cell proliferation.

Interstitial cell of Cajal loss correlates with the degree of inflammation in the human appendix and reverses after inflammation.

BACKGROUND: Normal gut motility relies on the complex interaction between the interstitial cell of Cajal (ICC) and the enteric nerve networks. Inflammation of the gastrointestinal tract adversely affects both ICC and enteric nerves. We aimed to determine the distribution of ICC and nerve networks in patients with appendicitis. METHODS: Specimens from controls and patients with appendicitis were examined with immunohistochemistry (c-Kit for ICC, beta III tubulin [Tuj-1] and neuronal nitric oxide synthase [histochemical diaphorase] for nitrergic neurons) and electron microscopy (EM). Data were quantified using image analysis. RESULTS: We found a profound decrease in c-Kit immunoreactivity (c-Kit IR) in the advanced inflammatory stages of appendicitis, which correlated with the severity of inflammation. Electron microscopy confirmed ultrastructural injury in both ICC and nerve fiber networks during acute inflammation. After the inflammation resolved, interval appendices displayed a recovery in ICC c-Kit IR to control levels and normal ultrastructure. The neuronal network also displayed ultrastructural recovery; however, neuronal nitric oxide synthase activity did not recover. CONCLUSIONS: Severe inflammation results in significant ultrastructural damage of nerves and ICC networks in appendicitis. The loss of c-Kit IR is likely due to impaired ICC cytophysiology because ICC was still present under EM. After resolution of acute inflammation, ICC recovers their normal ultrastructure and c-Kit IR.

Development of dissociated cryopreserved rat cortical neurons in vitro.

Dissociated neuronal cultures of various brain regions are commonly used to study physiological and pathophysiological processes in vitro. The data derived from these studies are often viewed to have relevance to processes taking place in the mature brain. However, due to the practical challenges associated with lengthy neuronal culture, neurons are often kept for 14 days in vitro (DIV), or less, before being subject to experimentation. Non-proliferative cultures such as primary neuronal cultures can be maintained for more than 42 DIV if water evaporation from culture media is monitored and corrected. To determine appropriate time points corresponding to the stages of cortical development, we compared characteristics of cryopreserved cortical neurons in cultures at various DIV using immunofluorescence, biochemical measurements and multielectrode array recordings. Compared to 21 and 35 DIV, at 14 DIV, cultures are still undergoing developmental changes and are not representative of adult in vivo brain tissue. Specifically, we noted significant lack in immunoreactivity for synaptic markers such as synapsin, vesicular GABA transporter and vesicular glutamate transporter at 14 DIV, relative to 21 and 35 DIV. Moreover, multielectrode array analysis indicated an increase in network firing up to 46 DIV with patterned firing peaking at 35 DIV. Our results provide specific evidence of the maturational stages of neurons in culture that can be used to more successfully plan various types of in vitro experimentation.

The corticostriatal system in dissociated cell culture.

The sparse connectivity within the striatum in vivo makes the investigation of individual corticostriatal synapses very difficult. Most studies of the corticostriatal input have been done using electrical stimulation under conditions where it is hard to identify the precise origin of the cortical input. We have employed an in vitro dissociated cell culture system that allows the identification of individual corticostriatal pairs and have been developing methods to study individual neuron inputs to striatal neurons. In mixed corticostriatal cultures, neurons had resting activity similar to the system in vivo. Up/down states were obvious and seemed to encompass the entire culture. Mixed cultures of cortical neurons from transgenic mice expressing green fluorescent protein with striatal neurons from wild-type mice of the same developmental stage allowed visual identification of individual candidate corticostriatal pairs. Recordings were performed between 12 and 37 days in vitro (DIV). To investigate synaptic connections we recorded from 69 corticostriatal pairs of which 44 were connected in one direction and 25 reciprocally. Of these connections 41 were corticostriatal (nine inhibitory) and 53 striatocortical (all inhibitory). The observed excitatory responses were of variable amplitude (-10 to -370 pA, n = 32). We found the connections very secure - with negligible failures on repeated stimulation (approximately 1 Hz) of the cortical neuron. Inhibitory corticostriatal responses were also observed (-13 to -314 pA, n = 9). Possibly due to the mixed type of culture we found an inhibitory striatocortical response (-14 to -598 pA, n = 53). We are now recording from neurons in separate compartments to more closely emulate neuroanatomical conditions but still with the possibility of the easier identification of the connectivity.

Cree antidiabetic plant extracts display mechanism-based inactivation of CYP3A4.

Seventeen Cree antidiabetic medicinal plants were studied to determine their potential to inhibit cytochrome P450 3A4 (CYP3A4) through mechanism-based inactivation (MBI). The ethanolic extracts of the medicinal plants were studied for their inhibition of CYP3A4 using the substrates testosterone and dibenzylfluorescein (DBF) in high pressure liquid chromatography (HPLC) and microtiter fluorometric assays, respectively. Using testosterone as a substrate, extracts of Alnus incana, Sarracenia purpurea, and Lycopodium clavatum were identified as potent CYP3A4 MBIs, while those from Abies balsamea, Picea mariana, Pinus banksiana, Rhododendron tomentosum, Kalmia angustifolia, and Picea glauca were identified as less potent inactivators. Not unexpectedly, the other substrate, DBF, showed a different profile of inhibition. Only A. balsamea was identified as a CYP3A4 MBI using DBF. Abies balsamea displayed both NADPH- and time-dependence of CYP3A4 inhibition using both substrates. Overall, several of the medicinal plants may markedly deplete CYP3A4 through MBI and, consequently, decrease the metabolism of CYP3A4 substrates including numerous medications used by diabetics.

GPI-1046 increases presenilin-1 expression and restores NMDA channel activity.

BACKGROUND: Previously we showed that 6-hydroxydopamine lesions of the substantia nigra eliminate corticostriatal LTP and that the neuroimmunolophilin ligand (NIL), GPI-1046, restores LTP. METHODS: We used cDNA microarrays to determine what mRNAs may be over- or under-expressed in response to lesioning and/or GPI-1046 treatment. Patch clamp recordings were performed to investigate changes in NMDA channel function before and after treatments. RESULTS: We found that 51 gene products were differentially expressed. Among these we found that GPI-1046 treatment up-regulated presenilin-1 (PS-1) mRNA abundance. This finding was confirmed using QPCR. PS-1 protein was also shown to be over-expressed in the striatum of lesioned/GPI-1046-treated rats. As PS-1 has been implicated in controlling NMDA-receptor function and LTP is reduced by lesioning we assayed NMDA mediated synaptic activity in striatal brain slices. The lesion-induced reduction of dopaminergic innervation was accompanied by the near complete loss of NDMA receptor-mediated synaptic transmission between the cortex and striatum. GPI-1046 treatment of the lesioned rats restored NMDA-mediated synaptic transmission but not the dopaminergic innervation. Restoration of NDMA channel function was apparently specific as the sodium channel current density was also reduced due to lesioning but GPI-1046 did not reverse this effect. We also found that restoration of NMDA receptor function was also not associated with either an increase in NMDA receptor mRNA or protein expression. CONCLUSION: As it has been previously shown that PS-1 is critical for normal NMDA receptor function, our data suggest that the improvement of excitatory neurotransmission occurs through the GPI-1046-induced up-regulation of PS-1.

Actions of ethnobotanically selected Cree anti-diabetic plants on human cytochrome P450 isoforms and flavin-containing monooxygenase 3.

AIM OF THE STUDY: Cree traditional medicine is commonly used concomitantly with prescribed drugs to treat health problems related to type II diabetes (T2D) that is endemic in the Cree population. However, the safety of traditional Cree medicines with respect to drug metabolism is unknown. MATERIALS AND METHODS: Seventeen anti-diabetic plant extracts were screened for their potential inhibition of 11 isoforms of the drug-metabolizing cytochrome P450s (CYPs), and flavin-containing monooxygenase 3 (FMO3) in fluorometric plate reader assays. Comparative analyses were conducted to determine if particular extracts were more inhibitory, or if particular enzymes were more inhibited. RESULTS: Many anti-diabetic plant extracts inhibited the CYPs, with CYP2C and 3A isoforms being most prone to inhibition. The order of inhibition for the enzymes by the Cree plant extracts was: 2C19>3A7>3A5>3A4>2C9>2C8>FMO3>1A2>2E1>19>2D6>2B6. Extracts from Rhododendron groenlandicum, Sorbus decora, and Kalmia angustifolia were identified as having strong inhibition towards many CYP isoforms. CONCLUSION: These findings demonstrate that extracts from most plant species examined have the potential to affect CYP2C- and 3A4-mediated metabolism, and have the potential to affect the bioavailability and pharmacokinetics of conventional and traditional medicines during concomitant use.

Colonic dysmotility in postsurgical patients with Hirschsprung's disease. Potential significance of abnormalities in the interstitial cells of Cajal and the enteric nervous system.

PURPOSE: Normal gut muscular function depends on the coordinated activity of both the enteric nervous system (ENS) and the interstitial cells of Cajal (ICC). Hirschsprung's disease (HD) has long been considered a purely neuronal deficit but recent data point to abnormalities in ICC in the proximal ganglionated HD colon. We examined the labeling of ICC and neuronal cells in the proximal ganglionated colon in patients with HD to determine whether abnormalities of ICC and ENS might be associated with a poor clinical outcome. METHODS: Tissue from 11 patients with HD was studied using immunohistochemistry for ICC and neuronal identification in comparison to control tissue from patients without HD. Image data were evaluated quantitatively and interpreted relative to clinical outcome. RESULTS: Interstitial cells of Cajal in the ganglionated colon of the HD group did not differ from the control group, but nerve cells/fibers were decreased 40%. Paired decreases in both nerve fibers and ICC in individual patients were associated with normal bowel function. Poor postoperative outcome was observed in a patient with normal innervation but with a profound decrease in ICC in the ganglionated colon. CONCLUSIONS: Nerve fibers are decreased in the proximal ganglionated colon in patients with HD without associated gut dysmotility. Poor clinical outcome was noted only in a patient with normal innervation and markedly decreased ICC. Collection of data from a much larger number of patients with poor clinical outcome will be necessary to determine the significance of this imbalance of ICC and innervation.

Secondary generalization of hippocampal kindled seizures in rats: examining the role of the piriform cortex.

A primary feature of epilepsy is the potential for focal seizures to recruit distant structures and generalize into convulsions. Key to understanding generalization is to identify critical structures facilitating the transition from focal to generalized seizures. In kindling, development of a primary site leads progressively to secondarily generalized convulsions. In addition, subsequent kindling of a secondary site results in rapid kindling from that site, presumably because of its facilitated access to the primary kindled network. Here, we investigated the role of the piriform cortex in convulsive generalization from a secondary site kindled in the hippocampus after primary site amygdala kindling. In a necessarily complicated design, rats initially experienced forebrain commissurotomy to lateralize the experiment to one hemisphere. Then the amygdala was kindled and, 3 weeks later, it was electrically-triggered into status epilepticus, which destroyed the ipsilateral piriform cortex. This experience occurred several days before secondary site kindling of the dorsal hippocampus. In rats with complete piriform cortex loss, there was no disruption in kindling or convulsive seizure expression from the hippocampus. However, when damage also involved parts of the perirhinal, insular and entorhinal cortices, convulsive expression was blocked. Although other evidence suggests that piriform lesions affect generalization of primary site kindling, the present study shows that they do not alter secondary site kindling in the dorsal hippocampus. The additional involvement of parahippocampal cortical areas in convulsive expression suggests an important functional association between these cortical regions and the hippocampus in seizure propagation and clinical expression.