PEDOTF8 were approximately 65% of the relative molecular weight values obtained by gel permeation chromatography using polystyrene standards. Both light absorption and molecular weight measurements showed the effective conjugation lengths . The low molecular weight polymers showed higher energy absorption and fluorescence bands. Molecular weight also affected the electrochemical process of the polymer films. The high molecular weight PEDOTF8 (number average molecular weight >70000) showed a higher redox stability than the low molecular weight one. The two polymers with number average molecular weights of 70200 and 40000 retained 65% and 25% of the charge storage capacity after 100 electrochemical scans, respectively. Square-wave potential step absorptiometry measurements of the polymers with number average molecular weights of 70200 and 40000 showed that the optical contrasts remain stable after ca. 55 and three cycles, respectively. The high molecular weight polymer has a high electrochemical stability and would be a good material for optoelectronic devices.Absolute molecular weight values obtained by gel permeation chromatography with multi angle light scattering of  EDOT, 3,4-ethylenedioxythiophene, is an attractive building unit for producing π-conjugated polymers4. The unit enhances the π-conjugation and p-doping through specific interactions and its excellent electron-donating property. Thus, PEDOT, poly, and EDOT-based alternating copolymers are high performance organic semiconductors and fluorophores4. For this reason, the focus is often placed on the optical (light absorption and fluorescence) and electrochemical (doping–dedoping) in the polymer film. Several parameters of the polymer affect its optoelectronic properties and device performance, such as the degree of polymerisation 7 and purity9. The molecular weight values of π-conjugated polymers play an important role in the performance of organic field effect transistors and organic solar cells7. The molecular weight values, including number average molecular weight (Mn) and weight average molecular weight (Mw), and polydispersity of the polymers are of central importance for their applications. Properties such as effective conjugation length and interpolymer coupling within the aggregates as well as the packing behaviour of the polymer chains affect the optoelectronic properties12. Low molecular weight π-conjugated polymers, which were insufficient effective conjugation lengths, offer higher energy light absorption (band gap) and fluorescence than high molecular weight ones14. Thus, low molecular weight fractions of the polydispersive polymers are critically affected to the optoelectronic properties.The optical and electronic properties of π-conjugated polymers are relevant for many applications in the field of semiconductors16. Because the direct arylation reactions have higher atom efficiency and reactivity than the other cross-coupling reactions18, the direct arylation polycondensation of EDOT with dibromoarenes have received great interest in recent years22. High molecular weight EDOT-based π-conjugated polymers (Mn > 80000) were successfully demonstrated by flow conditions19, microwave-assisted conditions20 and chloride-promoted conditions22. The molecular weight values of poly have been reported to affect light absorption properties19. Despite the investigation of this donor–acceptor polymer, the relationship between the molecular weight values and the light absorption properties of poly is still not fully understood. Poly shows a high FET hole mobility (1.2 × 10−3 cm2V−1s−1) and high OPV performance (PCE = 4%) depending on its molecular weight23. However, the effects of molecular weight on the fluorescence and electrochemical properties have not been investigated, which presents a challenging topic for understanding polymer properties. In this study, we investigate the light absorption, fluorescence and electrochemical properties of poly with different molecular weights.Polycondensation is a standard technique for synthesising the π-conjugated polymers. Synthetic pathways of EDOT-based π-conjugated polymers have been developed through palladium-catalysed cross-coupling researchEDOT with 2,7-dibromo-9,9-dioctylfluorene (F8) was performed at previously optimised conditions: 1.0 mol% of Pd catalyst (Pd(OAc)2, PdCl2, PITS-Cl)22, 30 mol% of 1-adamantanecarboxylic acid, (1AdCOOH) and 3.0 eq. of potassium carbonate (K2CO3) in dry N,N-dimethylacetamide (0.3 M) at 80–120 °C under argon for 5–120 min, giving poly, PEDOTF8, with different molecular weights . The Mn and Mw/Mn values of the polymers were 1800–95000 and 1.3–4.1, respectively , which are not the true (absolute) values because rigid (rod) segments of π-conjugated polymers imitate a large hydrodynamic radius that does not correspond to the molecular weight of polystyrene, which has coil segments. Thus, the average number of repeating unit (n) cannot be determined by the relative molecular weight from GPC. Moreover, calculating the n value by end-group determination using 1H NMR spectroscopy is difficult because the polymer was synthesised via polycondensation. Absolute molecular weight values of the polymers are required for determining n value. To estimate the values of absolute molecular weight, GPC with multi angle light scattering25 was performed (Table Mn and Mw) values of PEDOTF8 were approximately 65% of the relative molecular weight values  arylation polycondensation of ely Fig. . The meaues Fig. . This rePEDOTF8 polymers are displayed in Fig. PEDOTF8 in dichloromethane were shifted to a lower energy for increasing Mn values  showed similar absorption bands in both conditions  include fractions of insufficient effective conjugation lengths  material, the redshift of absorption band from solution to film is very small20. Compared with PEDOTF8 (Mn = 95000), PEDOTF8 (Mn = 1800) showed a large redshift from solution to film, indicating that the low molecular weight fractions in PEDOTF8 (Mn = 1800) form a domain for inter-polymer interaction  showed a low energy fluorescence band peaking at 515 nm (green-coloured fluorescence). However, the high molecular weight samples  showed fluorescence bands peaking at 549 nm (yellow-coloured fluorescence).Optical properties of the ues Fig. . The banues Fig. . The samons Fig. , displayths Fig. , right. ion Fig. . Fluoreshts Fig. . The lowPEDOTF8 was 2600. The number of EDOT-F8 units (n) of the MW was estimated at ca. 5 units. The fractions with an absolute molecular weight below 2600 (relative molecular weight <4000) and less than or equal to five EDOT-F8 units showed dramatically reduced optical properties  were polymers with insufficient effective conjugation lengths Fig. . The relies Fig. . Therefo26. Therefore, the film forming ability and toughness of amorphic PEDOTF8 depend on the molecular weight. Free-standing films of PEDOTF8 were prepared by casting PEDOTF8/toluene solution (10 g/L). The film of high molecular weight PEDOTF8 (Mn = 70200) showed tough films suitable for bending and rolling  produced brittle films.The film forming ability and toughness of polymers depend on the length of polymer segment (molecular weight) and crystalinitying Fig. . HoweverPEDOTF8 films on electrode evaluated the doping–dedoping process of counter ions. The process causes a volume change, destroying and detaching the films from the electrode or electrochemical quenching28. Reversible current responses of cyclic voltammograms indicate the high electrochemical stability (or toughness) of the films. A high molecular weight polymer shows toughness for volume change because of the entanglement among polymer segments. A three-electrode system equipped with the glassy carbon working electrode, platinum counter electrode and Ag/AgCl as a reference electrode was used in an electrolytic solution of 0.1 M Bu4NBF4/CH3CN. High molecular weight samples (Mn = 70200 and Mn = 95000) showed similar p- and n-doping–dedoping profiles at both oxidation and reduction  were different and showed irreversible n-doping–dedoping  of the polymer segment. Low molecular weight PEDOTF8 in film possesses higher content of the terminal groups than high molecular weight PEDOTF8.Cyclic voltammetry measurements of ion Fig. . Howevering Fig. . Moreoveing Fig. . The ele65% Fig.  and 25% 65% Fig.  of the cPEDOTF8 were examined by recording the UV-vis absorption spectra at different applied potentials. The drop-cast-coated thin films on an indium-tin-oxide-coated polyethylene terephthalate plate together with the reference Ag/AgCl and counter Pt electrodes were placed in a 1 cm cell. The UV-vis absorption spectra were measured in CH3CN containing 0.1 M (n-C4H9)4 NBF4 at 25 °C. The spectra for the polymer (Mn = 70200) film are illustrated in Fig. 32. The more oxidation in higher potential generally induces the new bands of bipolaron at different wavelength with decreasing of the band of polaron. Above a potential threshold of >1.0 V, the new bands were observed in the visible and near infrared regions around 600 and 980 nm, respectively, and the intensity at 700 nm decreased. The polymer film revealed a dark green colour in the fully oxidised state, because of the slight tailing of the absorption across the visible region.The spectroelectrochemical changes of PEDOTF8 (Mn = 70200) film exhibited long-term stabilities compared with low molecular weight PEDOTF8 films. The optical contrasts fall steadily at the initial stages and remain stable after ca. 55 repeated cycles  film were 8.1 and 4.9 s, respectively  film was slow for the colouring and bleaching processes  in the polymer affected the absorption and fluorescence properties. The high molecular weight PEDOTF8 samples showed a higher redox stability of the electrochemical doping–dedoping process of polymer films than the low molecular weight ones, which contributes to the development of a high performance electrochromic device. The molecular weight of the EDOT-based polymers is very important for optoelectronics.In conclusion, we successfully demonstrated the effect of molecular weight on the optical and electrochemical properties of 2CO3 (Kanto), palladium acetate (Wako), palladium chloride (Wako) and dry N,N-dimethylacetoamide (Wako) were used as received. PITS-Cl was prepared by the reported procedure22.3,4-Ethylenedioxythiophene (TCI Japan), 2,7-dibromo-9,9-dioctylfluorene (TCI Japan), 5,5′-dibromo-3,3′-dihexyl-2,2′-bithiophene (TCI Japan), 1-adamantanecarboxylic acid (TCI Japan), K2CO3  and 1.0 mol% of palladium (Pd(OAc)2, PdCl2, PITS-OAc, PITS-Cl) was stirred in dry N,N-dimethylacetoamide (1.0 mL) for 5–120 min at 80–120 °C under argon. The reaction mixture was diluted by toluene, rapidly cooled to room temperature, and then filtered to remove insoluble salts. The filtrate was poured into a large amount of methanol. The resulting polymer was collected by filtration and washed with a large amount of methanol. The polymer was dried under vacuum, producing an orange powder. The measurements of the polymer were performed without further treatment. The yield was estimated by the weight of the polymer, which was insoluble in methanol.A mixture of EDOT , 2,7-dibromo-9,9-dioctylfluorene , 1-adamantanecarboxylic acid , K1H NMR : δ 7.9–7.6 , 6.31 (end group of EDOT unit), 4.45 (O(CH2)2O, br), 2.05 (CH2(CH2)6CH3, br), 1.25–0.77 (CH2(CH2)6CH3, br). 13C NMR : δ 151.4, 139.5, 138.6, 131.7, 125.0, 120.3, 119.7, 116.1 64.6, 55.2, 40.4, 31.8, 30.0, 29.24, 29.21, 23.8, 22.6, 14.1. Anal. Calcd. for (C35H44O2S)n: C, 79.50; H, 8.39. Found: C, 79.28; H, 8.32.Yellow-coloured powder. 1H and 13C NMR spectra were recorded on a JEOL EX-300 spectrometer. Elemental analyses were performed on a Thermo Finnigan Flash EA1112 CHN-O analyser. GPC analyses were performed by a Toso GPC system (HLC-8220), using tetrahydrofuran as the eluent after calibration with polystyrene standards. GPC–MALS measurements were taken in THF at 25 °C (column temperature: 40 °C) using a Dawn EOS instrument  to evaluate absolute Mn and Mw of the polymers. The specific refractive index increment (qn/qc), which is necessary for the analysis of GPC with multi angle light scattering, was measured with an Otsuka Electric DRM-3000 (1⁄4633 nm) at 25 °C. The measured qn/qc of PEDOTF8 in THF was 0.5935 cm3 g−1. UV-vis absorption spectra were obtained on an Ocean Optics USB4000-XR1 fibre spectrometer with DH2000-BAL tungsten halogen light source. Fluorescence spectra were obtained on an Ocean Optics USB4000 fibre spectrometer with PX-2 pulsed xenon light source. Cyclic voltammetry measurements were performed by ALS 611. A three-electrode system equipped with the glassy carbon or indium-tin-oxide-coated polyethylene terephthalate electrode, platinum counter electrode and Ag/AgCl as a reference electrode was used in an electrolytic solution of acetonitrile containing 0.1 M tetraethylammonium tetrafluoroborate.Liquid-state


The transcription factor Nurr1 is essential for dopamine neuron differentiation and is important in maintaining dopamine synthesis and neurotransmission in the adult. Reduced Nurr1 function, due to the Nurr1-null heterozygous genotype (+/−), impacts dopamine neuron function in a region specific manner resulting in a decrease in dopamine synthesis in the dorsal and ventral striatum and a decrease in tissue dopamine levels in the ventral striatum. Additionally, maintenance of tissue dopamine levels in the dorsal striatum and survival of nigrostriatal dopamine neurons with aging (>15 months) or after various toxicant treatments are impaired. To further investigate the effects of aging and the Nurr1-null heterozygous genotype, we measured regional tissue dopamine levels, dopamine neuron numbers, body weight, open field activity and rota-rod performance in young (3–5 months) and aged (15–17 months) wild-type +/+ and +/− mice. Behavioral tests revealed no significant differences in rota-rod performance or basal open field activity as a result of aging or genotype. The +/− mice did show a significant increase in open field activity after 3 min of restraint stress. No differences in tissue dopamine levels were found in the dorsal striatum. However, there were significant reductions in tissue dopamine levels in the ventral striatum, which was separated into the nucleus accumbens core and shell, in the aged +/− mice. These data indicate that the mesoaccumbens system is more susceptible to the combination of aging and the +/− genotype than the nigrostriatal system. Additionally, the effects of aging and the +/− genotype may be dependent on genetic background or housing conditions. As Nurr1 mutations have been implicated in a number of diseases associated with dopamine neurotransmission, further data is needed to understand why and how Nurr1 can have differential functions across different dopamine neuron populations in aging. Dopamine neurotransmission has been implicated in a number of pathological conditions including Parkinson’s disease, schizophrenia, attention deficit hyperactivity disorder and addiction –4. Nurr1In vitro and in vivo studies demonstrate that Nurr1 gene delivery/therapy and Nurr1 activation/activating compounds enhance DA as well as protect mesencehpalic dopaminergic neurons from cell injury induced by toxins or neuroinflammation and nigrostraiatal associated motor behaviors of dopamine neurotransmission [Several intrinsic mechanisms have been identified in the mesencephalic DA neurons that are linked to Nurr1 mediated cell survival –14. In vsmission –18. Nurrsmission ,20.Mutation analysis has implicated a role for Nurr1 in some of these pathological conditions. Mutations in Nurr1 have been linked to Parkinson’s disease –24 and NNurr1 has been implicated as a transcription factor that regulates the expression of several dopamine neurotransmission genes including tyrosine hydroxylase, dopamine transporter, vesicular monoamine transporter and GTP cyclohydrolase ,26,29–33Quinpirole, standards for high performance liquid chromatography (HPLC) analysis including dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and HPLC reagents were purchased from Sigma- Aldrich . CNS perfusion fluid was purchased from CMA Microdialysis . Reagents for the HPLC mobile phase were purchased from Sigma-Aldrich .ad libitum. Care of the mice was overseen by a laboratory animal veterinarian. Male mice were used for behavior analysis, immunohistochemistry, and neurochemistry measurements.The Nurr1-null heterozygous mice used for this study were obtained from a colony bred at Mississippi State University originally produced in the laboratory of Dr. Vera Nikodem at the National Institute for Diabetes and Digestive and Kidney Diseases . Mice weTo assess motor coordination, mice were tested using a 4 station rota-rod treadmill for mice . The rota-rod was set to increase rotation speed from 3–30 rpm. Each mouse underwent 2 rounds of training. The mice were placed on the rod for 2 min. If the mouse fell off during this time it was placed back on the treadmill so that all mice received the same amount of training time. Each mouse had a rest time 4 min in between each training round. After the training, 3 test trials were done on each mouse. The time spent and the speed reached by each mouse was recorded and compared between +/+ and +/− young and aged mice. At the end of rota-rod testing, mice were weighed. To assess spontaneous and stress induced locomotor activity, mice were placed in an open field chamber consisted of a 25 cm × 25 cm plexiglass enclosure with a video camera mounted above and attached to a computer containing the LimeLight software to measure total distance traveled. The basal activity of the mice is monitored initially for 45 min by a video camera. After this 45 min activity period, mice were placed in a Broome rodent restrainer for 3 min to produce restraint stress, then the mice were put back into the open field chamber and the activity was monitored for another 45 min. The total distance traveled during the basal condition and the stress condition was compared across age and genotype. The number of mice used for behavior analysis included 22 +/+ young, 37 +/− young, 7 +/+ aged, and 8 +/− aged.2 asphyxiation, the brains removed, cut with a coronal section at approximately 1 mm caudal to Bregma into forebrain and midbrain pieces. The forebrain was further split in half with a sagittal cut. The midbrain piece and the right forebrain piece were immersion fixed in 4% paraformaldehyde for 24 h then placed in 30% sucrose for 2 days. The left forebrain piece was frozen on dry ice and stored at −80°C.One week after the open field test, the mice were euthanized with COThe left piece of frozen forebrain tissue was mounted in a custom made tissue slicer with OCT compound  and 600–800 µm frozen sections were cut and mounted on glass slides. Micropunches of the dorsal striatum were isolated using a blunt 20 gauge needle. Micropunches of the nucleus accumbens core was taken with a 22 gauge blunt needle then the remaining nucleus accumbens shell was dissected using an 18 gauge blunt needle. A micropunch of the prefrontal cortex was also taken with a blunt 20 gauge needle. Approximate locations of these dissected regions are shown on tyrosine hydroxylase immunohistochemistry sections in Tissue extractions (10–20 µL) or microdialysis fractions (18 µL) were injected into a HPLC system consisting of a Waters 2695 Separation module, a SupleCosil LC-18-DB column with the Waters 2465 electrochemical detector set at 20 nA and an Ec=+0.67 V using a mobile phase of 100 mM phosphate, 17.5% methanol, 25 µM EDTA, 1 mM octyl sodium sulfate at pH 3.65. The quantity of each compound was determined based on the response of a known amount of standards of dopamine, DOPAC and HVA  and are reported as pg in the dialysate.2O2 in PBS. Sections were washed three times in PBS then incubated in blocking serum  for 30 min. Sections were then incubated in a rabbit polyclonal anti-tyrosine hydroxylase antibody  diluted 1:5000 at room temperature for 2 h. Sections were rinsed 10 times with PBS containing 1% BSA and 0.2% Triton X-100 then incubated in biotinylated anti-rabbit IgG for 2 hours, rinsed 3 times. Sections were then incubated for 2 h in equilibrated ABC reagent  diluted in PBS-0.02% Triton X-100 and 1.0% bovine serum albumin. Sections were rinsed 2 times in PBS then incubated in a 0.5× diaminobenzidine solution  with 0.003% H2O2 for 5 min. Sections were rinsed in PBS and counterstained with nuclear fast red . Sections were mounted onto silanized slides, dehydrated in graded ethanol series followed by xylene, then cover slipped with Permount. Immunoreactivity was evaluated and stereology was performed using an Olympus BX51 microscope with a CCD camera and a motorized Z-stage which is all connected to a computer with Stereo Investigator Stereology Software from MicroBrightField Inc. . Unbiased stereology on tyrosine hydroxylase immunoreactive profiles in the substantia nigra pars compacta and ventral tegmental area was performed using Stereo Investigator Stereology Software from MicroBrightField Inc. The optical fractionation method was used for estimating tyrosine hydroxylase immunoreactive profiles. The substantia nigra pars compacta and the ventral tegmental area were outlined based on tyrosine hydroxylase labeling. A 60× oil immersion objective was used to count profiles and measure section thickness at each sample site. Stereologic parameters used were a grid size of 150 µm × 150 µm with a random orientation and an optical dissector height of 16 µm. Estimates of immunoreactive profiles were made in young and aged +/+ and +/− mice (n=3/group) in both the substantia nigra pars compacta and ventral tegmental area. The number of mice used for stereological counts consisted of 3 +/+ young, 3 +/− young, 3 +/+ aged, and 3 +/− aged.Fixed midbrain and forebrain tissue was serial sectioned into 30 nm sections. Every 6th section was used for immunhistochemistry. Free-floating sections were washed 3 times in phosphate buffered saline (PBS) with 1% bovine serum albumin (BSA), and then incubated for 30 min in 1% HBehavioral data, tissue catecholamine levels and stereological estimates were analyzed using ANOVA with Fisher’s PLSD post-hoc comparison.Behavioral analysis was carried out on the young and aged, +/+ and +/− mice. The behavioral test consisted of rota-rod performance, open field activity in a novel environment and after 3 min of restraint stress. The weight of each mouse was also recorded. Aging significantly increased weight. Although there was no significant difference across genotype, the +/− mice had slightly lower body weights . There wTissue dopamine and metabolite levels were measured in micropunches from the dorsal striatum, the ventral striatum separated into the NAC and NAS, and the prefrontal cortex across young and aged +/+ and +/− mice. Within the dorsal striatum, there were no significant differences in tissue dopamine levels observed due to the +/− genotype or aging  . In factSurvival and innervations of dopamine neurons was determined across aging and the +/− genotype using tyrosine hydroxylase immunohistochemistry. To determine if aging and the +/− genotype affected survival of dopamine neurons, unbiased stereology was used to estimate dopamine neuron population in the substantia nigra pars compacta and the ventral tegmental area. No noticeable differences in the intensity or distribution of dopamine neurons in either the substantia nigra pars compacts or ventral tegmental area were observed across groups in either of these areas . UnbiaseCurrently, animal models that reproduce all of the neuropathology and neurodegeneration found in Parkinson’s disease are lacking. The most widely used models consist of the use of various toxins to kill or damage dopamine neurons. Because of the important contribution of aging in the etiology of Parkinson’s disease as well as links to genetics, a more ideal model would consist of a genetic mutation that, when combined with aging, produces progressive deficits in dopamine levels and neurodegeneration of nigrostriatal dopamine neurons. Previous reports using aged Nurr1 +/− mice have described significant effects in the nigrostriatal dopamine system. Aging (as early as 9–12 months) in the Nurr1 +/− mice resulted in a significant reduction in dopamine levels in the striatum, impaired performance on the rota-rod, and reduced numbers of dopamine neurons in the substantia nigra pars compacta in the aged +/− mice ,43. BecaThe Nurr1 +/− genotype appears to be an important regulator of tissue dopamine as it relates to the mesoaccumbens dopamine system and that aging is an important variable in how Nurr1 regulates dopamine levels. As for the nigrostriatal dopamine system, there are, apparently, other factors that influence whether dopamine neuron numbers and tissue dopamine levels are affected by the +/− genotype. Understanding the interaction between how the environment or genetic background can interact with the Nurr1 +/− genotype could have important implications for understanding the genetic complexity of Parkinson’s disease. The interaction between aging and the +/− genotype suggest that aging is an important factor for the regulation of Nurr1 and the function of the mesoaccumbens dopamine system, which could have implications to other neurological problems such as psychosis, addiction or attention deficit hyperactivity disorder in which the mesoaccumbens dopamine neurotransmission has a prominent role.


The evolution of cellular pathology as a specialty has always been driven by technological developments and the clinical relevance of incorporating novel investigations into diagnostic practice. In recent years, the molecular characterisation of cancer has become of crucial relevance in patient treatment both for predictive testing and subclassification of certain tumours. Much of this has become possible due to the availability of next-generation sequencing technologies and the whole-genome sequencing of tumours is now being rolled out into clinical practice in England via the 100 000 Genome Project. The effective integration of cellular pathology reporting and genomic characterisation is crucial to ensure the morphological and genomic data are interpreted in the relevant context, though despite this, in many UK centres molecular testing is entirely detached from cellular pathology departments. The CM-Path initiative recognises there is a genomics knowledge and skills gap within cellular pathology that needs to be bridged through an upskilling of the current workforce and a redesign of pathology training. Bridging this gap will allow the development of an integrated ‘morphomolecular pathology’ specialty, which can maintain the relevance of cellular pathology at the centre of cancer patient management and allow the pathology community to continue to be a major influence in cancer discovery as well as playing a driving role in the delivery of precision medicine approaches. Here, several alternative models of pathology training, designed to address this challenge, are presented and appraised. The subject of pathology has been in evolution for over 2000 years, with progress made following waves of intellectual and technological advancement. Early pathological understanding arose from the clinical descriptions of infections, inflammation and tumours first described by Hippocrates. Renaissance-era physicians and anatomists developed this understanding by describing gross pathological appearances seen through human dissection, and later Thomas Hodgkin was the first physician to define pathology by the microscopic appearances in human tissue, including the disease that now bears his name.The extensive work of Rudolf Virchow, by many regarded as the greatest figure in the history of pathology, in describing and categorising human disease by cellular appearances layed the basis for the development of pathology as both a clinical discipline and a modern science. In the late 19th and early 20th centuries, teachers in morbid anatomy were found in medical schools throughout Europe representing the early ‘academic pathologists’. Meanwhile, the clinical practice of ‘surgical pathology’ was beginning to be performed by physicians and surgeons who used microscopic findings for clinical diagnosis. By the mid-20th century, these roles were consolidated as histopathology became recognised as a medical diagnostic discipline in its own right, one based on light microscopy, and with a strong academic ethos.In 1998, 45 years after the discovery of the DNA helix, the director of the National Cancer Institute challenged the scientific community to ‘harness the power of comprehensive molecular analysis technologies to make the classification of tumors vastly more informative’ and to change ‘the basis of tumor classification from morphological to molecular characteristics’.Traditional pathology training has taught us to recognise and distinguish a myriad of pathological entities based on their macroscopic and microscopic morphological appearances. Complex diagnoses are founded on years of collective experience and an element of subjectivity. This is in contrast with molecular diagnostic methods, which are heavily technology driven and produce objective and quantitative data. Immunohistochemistry (IHC) sits somewhere between the two.While IHC has been adopted into routine practice and pathology training, in many institutions ‘traditional’ morphological pathology and ‘new molecular’ pathology laboratories exist in parallel, with little interaction. A notable exception to this is the work of the Specialist Integrated Haematological Malignancy Diagnostic Service (SIHMDS) in the UK. SIHMDS services combine elements of laboratory haematology, histopathology, flow cytometry, cytogenetics and molecular medicine prior to the production of a finalised integrated report, which permits internal validation and crosschecking. The development of this model has not been without its difficulties: centralisation of services, co-location of several laboratories and information technology and computing infrastructure. Cellular pathology has much to learn, both from the successes and difficulties that SIHMDS has experienced.There are currently a number of tissue-based molecular tests used in routine diagnostic practice, although the exposure of individual pathologists will be dependent on their area of practice, as in some subspecialties, molecular diagnostics has progressed more rapidly than in others.As our understanding of cancer biology progresses and new technologies develop, extension of these single-gene tests or low-target tests has the potential to completely reshape the field of tissue-based and cell-based molecular testing.IHC already has an established role in the histopathological taxonomy of diseases, with disease-specific ‘patterns of immunohistochemical expression’ now a standard component of a pathologist’s diagnostic acumen. In recent decades, new uses have been postulated for some of these immunohistochemical biomarkers. For instance, the presence (or absence) of expression of a protein may be perceived as evidence of an inherited disease. In the field of cancer immunology, the assessment of markers of adaptive immunity  and immune checkpoints  show promise as prognostic and predictive indicators. Indeed, in the era of personalised medicine, immunohistochemical expression may dictate a certain therapeutic intervention.With the notable exceptions of haematological malignancies and more recently brain tumours, the mainstay of cancer classification is morphology. However, large-scale genomic profiling studies have demonstrated the level of tumour diversity at a genomic level: genomic and transcriptomic analysis of breast cancer identified 10 ‘integrative subtypes’ with prognostic and therapeutic implications,High-throughput genomic profiling makes possible rapid and comprehensive analysis of cancer tissues even from small biopsy samples. Predictive (diagnostic) biomarkers can be used to match patients with targeted therapies. Prognostic markers can be integrated into existing clinical staging systems for improved patient stratification. However, in spite of the large number of credible biomarkers identified in basic research studies, relatively few have been validated and accepted for routine clinical use. The steps to the translation of biomarkers into clinical use are summarised in The level of intratumour and intertumour heterogeneityThe impact of molecular technologies on patient assessment, diagnosis and management means the role of the pathologist will expand and evolve. Apart from traditional morphological assessment of tissues and cells, and accompanying molecular tests, pathology is at the interface between biomarker validation, biomarker adoption and diagnostics.Pathologists have a key role in the integration of molecular medicine into therapeutic pathways through their influence in biomarker translation, clinical trials and clinical data systems.Much of the large failure rate in the lack of clinical applicability of biomarker studies is in the fact that validation and biomarker adoption are suboptimally executed.Pathologists supporting clinical trials in local hospitals, and leading the pathology for multicentre clinical trials, require knowledge of the design and governance of trials, familiarity with laboratory protocols and an understanding of the analytical and biobanking aspects of trial management. This has relevance for both trials requiring patient stratification (close to a diagnostic activity) and those aiming to discover new determinants of response.The management of pathological information in the context of LIMS (laboratory information management systems) and its integration in the context of hospital information systems will improve the quality of the diagnostic service. Equally, the use of this information for research purposes, within the requirements of anonymity, confidentiality and ethical approval, will facilitate hospital-based discovery.The development of digital pathology, pathology bioinformatics and biobanking represent critical areas of pathology activity in scientific discovery. Digital pathology and the adoption of image analysis have grown rapidly in the last few years due to advances in software, hardware and computer processing capacity, as well as the increasing importance of tissue-based research for biomarker discovery and stratified medicine. Digital pathology and image analysis have important roles across the drug/companion diagnostic development pipeline and in molecular pathology studies, tissue microarray (TMA) analysis, biobanking and molecular profiling of tissue. Integrating digital image analysis data with epidemiological, clinical and genomic data will help maximise the potential of tissue samples in delivering personalised medicine.The analysis and management of large genomics data will be at the heart of the delivery of the promise of modern genomic medicine. Effective integration of morphology with the ample available genomic information will determine the value of pathology in the future.Repositories containing high-quality human biospecimens linked with robust and relevant clinical and pathological information are required for the discovery and validation of biomarkers for disease diagnosis, progression and response to treatment.Both Pathology and Epidemiology aim to elucidate the aetiology of disease and the integrated discipline of ‘molecular pathological epidemiology’ aims to interrogate large sample cohorts from epidemiological studies with pathological tools such as IHC and TMAs, digital image analysis and high-throughput genomics, and link with epidemiological data to reach significant and practical discoveries.In recent decades, pathology has witnessed a separation of the academic and diagnostic, to the extent that many Pathology departments in leading UK teaching hospitals lack any substantial academic activity. Activity to realign the diagnostic and research agendas, with specific models to do so, is gaining traction in the UK. The aim is to draw technological expertise from the research environment and to bring a culture of accreditation and rigour from diagnostics into research.The aim of this document is to propose key points of considerations for training the next generation of pathologists and pathology leaders, equipped to deliver healthcare founded on morphomolecular diagnoses. But how much molecular pathology should a diagnostic pathologist know?It is a common opinion that the diagnostic histopathologist should integrate morphological and molecular information. There are two types of integration to consider: diagnostic and therapeutic. Diagnostic integration is exemplified by the haemato-oncology integrated report, where the result of clonality testing or the presence of a translocation can confirm/refute a diagnosis of malignancy and provide a clear diagnostic certainty. This paradigm is shared by, at least, neuropathology and sarcoma/soft tissue pathology.an understanding of the technologies that generated the results;judgement as to whether a specific test is technically satisfactory with the ability to troubleshoot a suboptimal test run;an understanding of the biological variables that some of these tests may bring to the forefront in some cases;knowledge to manage increasingly complex data ;an understanding of the clinical relevance of test results.Therapeutic integration informs a therapeutic decision. This requires very broad knowledge and specific training in molecular diagnostics. It requires:At the moment, pathology training does not deliver these key skills and recent attempts to redefine training, while admirable, remain too limited. Training programmes that can produce pathologists who are also proficient in molecular diagnostics need to be developed, although there are significant challenges in developing such a programme; training as a competent morphologist in most areas of pathology requires a full 5 years of training, there are very few molecular pathology centres with the capacity to train a steady stream of molecular pathologists and the process for modifying a curriculum through the General Medical Council (GMC) is complex and lengthy. Here, we consider three potential models of delivering a future pathology workforce in the UK, taking as a baseline the current training structured championed by the Royal College of Pathologists.Model 1 would aim to deliver morphomolecular training within the current 5-year training timeframe. This would require some condensation of the current morphological training, perhaps by allowing autopsy or gynae cytopathology to be dropped after stage A of training, and delivering a select amount of molecular training. This would include molecular pathology lectures in the early stages of training, followed by a compulsory 2-month to 3-month molecular attachment and an optional final year training in molecular diagnostics. Overall, this represents a meaningful increase in the molecular diagnostic training within the existing timeframe and structure of general histopathology training. Early training in the module has the capacity to be delivered in national/regional blocks, and there is the potential for a distance learning component in the pre-FRCPath Part 1 phase. This increase in molecular diagnostic training would need to be matched by an equally significant examined component of the FRCPath examinations. This model has the advantage of delivering a basic level of molecular pathology training to all, with a view to delivering an entire workforce with background in molecular diagnostics and the flexibility in stage D for suitably interested trainees to develop these skills further. Delivering this model of training within the currently existing 5-year RCPath histopathology training can be done in the appropriate centre,Model 2 focuses the delivery of molecular pathology training within an optional extension of training to allow an MSc or Fellowship in Molecular Pathology to be undertaken before or after the FRCPath Part 2. A plethora of Molecular Pathology MScs are currently available in the UK through which this additional training could be delivered to the relevant trainees. As the MSc programmes are optional, this could be perceived as an additional burden beyond the already demanding requirements of histopathology training, however, and if undersubscribed by pathology trainees, molecular pathology MSc programmes may fail to address the skills gap in molecular pathology. It is crucial that if these programmes aim to upskill trainees in molecular diagnostic pathology, the projects offered allow development of expertise in this area and are not predominantly focused on delivering basic science projects, which are used primarily as a segue into academic training.Model 3 would involve targeted centres, which show they have the capacity to train morphomolecular pathologists, to start doing so, in programmes accredited by the RCPath, in parallel to conventional histopathology training. This would be delivered within the context of a modular training programme combining morphological and molecular training and would follow an approved RCPath template. It is envisaged that while this ‘hybrid’ morphomolecular training programme is delivered in selected centres, the majority would continue with the standard training currently being delivered to histopathology trainees. These parallel training programmes would potentially reflect more accurately the true distinction between general pathologists, who require the balanced general training to report a wide range of specimens, completing training the current RCPath curriculum (or proposed model 1), and pathologists who practice monospecialist reporting in centres that also deliver molecular pathology testing being trained through model 3.Molecular pathology is a vast area that is constantly evolving, and it is difficult to have a grasp of every technique and application and to learn about techniques in an abstract manner. It would therefore be useful to link molecular techniques with the context of a pathology subspecialty within a modular training programme. This proposal will prove some logistical challenges to the relevant training centres and the RCPath, although the model would not be unlike the specialist training delivered and examined after stage B in head and neck pathology for dentally qualified pathologists.Due to the national strategic importance of genomic medicine within the UK and the already problematic skills gap in molecular pathology, such morphomolecular training posts should be considered additional to the existing histopathology training numbers with a national-level funding model that reflects this.The discipline of histopathology is at the crossroads. To maintain its central role in the evaluation of patients, pathologists will need to fully incorporate molecular diagnostics for the understanding and characterisation of diseases. If surgical pathologists attempt, even passively, to resist this change, this is likely to invite others to perform molecular testing of surgical pathology samples, moving the central role of tissue-based diagnosis out of the field of pathology. Crucially, this will be a poor outcome for patients, as morphological and molecular knowledge that needs to be integrated will be fragmented into different specialities. The strategic position of our discipline at the centre of clinical practice may be lost, reducing the importance of histopathology as a clinical specialty.The resulting marginalisation of histopathology in molecular medicine will further diminish the current strategic position of histopathology at the interface between clinical medicine and biomedical research. Failure to learn the language of molecular biology will significantly reduce the input and engagement from histopathologists in tissue-based research and their valuable skills will become a more remote resource to the biomedical research community, which will ultimately impact negatively on the quality of research. In addition, molecular diagnostics is a rapidly growing area of medicine, moving a budget of many billions of dollars, and appropriately trained molecular pathologists are potentially best placed to make appropriate decisions about how these considerable investments are made to support optimal, combined morphomolecular diagnostics. Histopathologists’ disengagement from molecular medicine threatens to exclude our departments from significant potential sources of revenue.The CM-Path group authoring this document has appraised the options identified for training the next generation of histopathologists. The group believes that molecular diagnostic training should represent a substantial component of the histopathology curriculum in the UK, far beyond what is currently delivered, although it is understood that the Royal College of Pathologists’ position is that there is little margin to change the curriculum at this time.A revisited version of today’s curriculum, with a significant increase in molecular diagnostic training, taking place within the current 5-year programme (described in model 1) and increasing the time devoted to the theory and practice of molecular pathology is certainly the minimum change that the group feels is required to properly equip the specialty for future pathology practice. Model 2 of training has potential as an interim solution while more radical change in the curriculum is developed although the optional and variable nature of the molecular pathology component makes this an inadequate long-term plan for upskilling the profession.Overall, the CM-Path group considers that, in order to secure the future of the specialty at the fulcrum of modern medicine, a parallel training programme should be implemented. This would include one pathway based on current training, which it is envisaged the vast majority will follow in the first instance, and an alternate option of incorporating in-depth molecular diagnostic training over the 5 years by developing modular morphomolecular pathology training programmes (model 3). This parallel subspecialist pathway will develop a cohort of highly skilled molecular histopathologists both able to strategically secure tissue-based molecular diagnostics within our specialty and capable of embracing the inevitable increase in morphomolecular diagnostic complexity that each subspecialty will face over the coming years.It is our view that maintaining the status quo will be detrimental for both the next generation of pathologists and for the specialty as a whole. Whether or not we embrace radical change, the eventual future of histopathology will be our responsibility. Do we need to nurture molecular pathology as much as morphology? How much of the revolution in molecular medicine do we wish to embrace? These are questions that our generation can ask itself, though if we do not act accordingly, we may be the last generation of pathologists able to do so.Molecular diagnostics is one of the fastest growing areas of medicine.The involvement of pathology in the modern taxonomy of disease has become a matter of general importance for the medical and scientific community.Pathology and pathologists need to rise to the challenge of genomic medicine, this will dictate the extent and quality of genomic medicine over the next decade.The National Cancer Research Institute’s CM-Path initiative would like to see a change in the postgraduate pathology curriculum, to create a new morphomolecular pathology specialty in order to deliver high quality molecular diagnostics for patient benefit.


Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells.Functional interactions in signaling occur between dopamine D


Bisdermican derived from subconfluent cultures of fetal fibroblasts displayed the largest glycosaminoglycan chains with a molecular weight of 31.5 kDa for the large subunit, and a molecular weight of 22 kDa for the small subunit. Thus, Bisdermican displays a molecular polymorphism that is related to its chronological age and proliferative state.Bisdermican (PG760) is a large, heterodimeric, dermatan sulfate proteoglycan found in selected basement membranes, smooth muscle cell layers, and different extracellular matrices. Age-dependent and developmentally regulated alterations in glycosaminoglycan structure and quantity have been shown to be functionally relevant for a number of physiological and pathological processes. Bisdermican was purified from human skin fibroblast cultures of different age and confluency. Following β-elimination, glycosaminoglycan chains were analyzed by Sephacryl-S-300 chromatography. Glycosaminoglycan chains of Bisdermican from infantile fibroblasts had a molecular weight of 19 kDa, whereas the glycosaminoglycan chain of the large Bisdermican subunit purified from confluent fetal fibroblast secretions was slightly larger (M


Despite several inconsistencies and methodological biases , the dopJauhar et al.  includedcause psychosis (or proneness to psychosis). Howes and Kapur  is approximately 75 times less than risperidone and 100 times less than haloperidol (2 dopamine blocking properties of different antipsychotics revealed clozapine to be an outlier (There are two lines of evidence that challenge the DH. First, according to a comprehensive meta-analysis of randomized trials, antipsychotic medication does not prevent the development of schizophrenia in persons at ultra-high risk of psychosis . If incroperidol . An anal outlier . Yet clo outlier  if not m outlier  than othThe commonly conducted experimental tests of the DH, such as the F-dopa uptake studies, are inconclusive due to several limitations. Larger sample sizes with antipsychotic naïve participants, adequate control of possible confounders and stringent comparators are necessary to provide a convincing test of the DH. If possible, researchers should measure striatal dopamine concentration before and after the onset of psychosis and statistically control for important covariates such as a person's activity, diet, and substance use, which impact on various neurobiological systems besides dopamine. Future work should also examine how stress, with its complex and multi-factorial brain mechanisms, may account for both striatal dopamine concentration and the experience of psychotic symptoms. Finally, the limitations outlined in this opinion paper also apply to other targets of drug action. Simply shifting the focus to another isolated neurotransmitter, such as for instance histamine, and analyzing its action in complete disregard of interactions between neurochemicals, complex neurological signaling networks and functional feedback loops, will not sufficiently advance our knowledge of the multiple, inter-related neurobiological pathways underlying psychosis.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.JM is a member and co-chair of the Critical Psychiatry Network. The other author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The neonatal Fc receptor (FcRn) plays a crucial role in transporting IgG and associated antigens across polarized epithelial barriers in mucosal immunity. However, it was not clear that FcRn expression in aggregated lymphoid nodules area (ALNA) in abomasum, a unique and important mucosal immune structure discovered only in Bactrian camels. In the present study, 27 Alashan Bactrian camels were divided into the following five age groups: fetus (10–13 months of gestation), young (1–2 years), pubertal (3–5 years), middle-aged (6–16 years) and old (17–20 years). The FcRn expressions were observed and analyzed in detail with histology, immunohistochemistry, micro-image analysis and statistical methods.P < 0.05). In addition, the FcRn was also expressed in the vessel endothelium, smooth muscle tissue, and macrophages and dendritic cells (DCs) of secondary lymphoid follicles (sLFs).The results showed that the FcRn was expressed in mucosal epithelial cells of ALNA from the fetus to the old group, although the expression level rapidly declined in old group; moreover, after the ALNA maturated, the FcRn expression level in the non-follicle-associated epithelium (non-FAE) was significantly higher than that in FAE  could participate in mucosal immune response and tolerance in ALNA of Bactrian camels through FcRn transmembrane transport. In mucosal immunity, polymeric immunoglobulin receptor (pIgR) is an important receptor, which helps to maintain mucosal barrier integrity and gastroenteric homeostasis by transporting secretory immunoglobulin A (SIgA) antibodies across intestinal epithelial cells (IECs) into gut secretions –5. As weFcRn, originally discovered in the intestinal epithelium of newborn rat , is alsoBactrian camel is an important livestock of economic characteristics in northwest of China and has some special immunological features. First, compared with the structure of conventional IgG, the Camelidae IgG2 and IgG3 are special heavy chain antibodies (HCAbs) , which an = 3, two males and one female), young , pubertal , middle-aged  and old . Fetus tissues were collected from animal carcasses submitted to the necropsy service in College of Veterinary Medicine, Gansu Agricultural University. Other animals were from the slaughterhouse  and were not starved before slaughter, which were anaesthetised intravenously with sodium pentobarbital (20 mg/kg) and then exsanguinated to death.Twenty-seven Alashan Bactrian camels of different ages  were divided into five age groups: fetus . For blocking, 5 % bovine serum albumin  was used. All samples with the primary antibody were incubated at 4 °C overnight. After being rinsed with PBS 2 min × 3 times; HRP conjugated goat anti-rabbit IgG  as secondary antibody was applied for 1 h in humidified box at 37 °C. After being rinsed with PBS 5 min × 4 times. The SABC was applied for 30 min in humidified box at 37 °C. After being rinsed with PBS 5 min × 4 times. For detection, DAB Kit  was used at room temperature. Slides were counterstained with Hematoxylin  and mounted with Neutral Balsam . Sections were examined with an Olympus microscope  [The whole abomasum from the isthmus to the pyloric ostium was incised along the greater curvature. The gastric contents were cleaned with saline. Samples from RMFR and LMFR of ANLNA were rapidly taken after death and fixed in 10 % neutral formalin. The paraffin sections were made and stained with haematoxylin and eosin (H&E) by a routine method  as well Germany) .Rabbit polyclonal antibodies against human FcRn, diluted with the buffer at 3.33 μg/ml before use, was supported by BIOSS .+ T cells, and the antigen peptide should be composed of more than 12 amino acid residues. The molecular weight of human FcRn is about 50 kDa. Moreover, the similarity of FcRn between dromedary and human was 78.6 % by analyzing the genes of phylogenetic relatedness of the extracellular domains (α1–α2–α3) [Some studies reported that the drFcRn/ Fc contact residues were highly conserved, and the structures of FcRn in different species were similar , 34, 43.SABC goat anti-rabbit IgG polyclonal antibodies immunohistochemical kit . The kit contained 1.5% BSA:12 ml. Second antibodies: Biotin goat anti rabbit IgG: 12 ml. SABC: 12 ml. It is an easy-to-use kit, which can be used directly and is unnecessary to be diluted with the buffer.In each group, the expression sites and characteristics of FcRn were observed in detail under microscope and photomicrographed using Olympus DP-71 microscopy system.P < 0.05.Five sections were randomly selected for each sample. Ten microscopic fields were randomly selected, observed and photomicrographed for FAE, non-FAE, vascular, smooth muscle and lymphoid follicle in each section. The mean optical density (MOD) of each site was calculated (Image-Pro Plus 6.0), respectively. The main steps contain: 1. the background correction of the IHC photos ; 2. the correction of the optical density of the IHC photos ; 3. the parameter setting  as the measurement value of the ICH image); 4. selection the measurement region through the software tools; 5. color settings; 6. measurement of the IOD and the area of the selected region; 7. the calculation of the MOD through IOD/selected areas. The MOD differences among groups were analyzed by one-way ANOVA followed by Duncan’s new multiple range test using IBM SPSS v. 23.0 . The significant difference was considered at (1') In RMFR, a plenty of primary lymphatic follicles (pLFs) were primary densely-distributed in the lamina propria (LP)  In LMFR, the histological characteristics were similar to those in RMFR. Lymphatic follicles were also pLFs. But the distribution density was lower than that in RMFR  Fig.  and FAE LP) Fig. , and maiLP) Fig. . In addiLP) Fig.  and smooLP) Fig. . (2') InMFR Fig. . The locMFR Fig. .Fig. 1Hi(1') In RMFR, a plenty of secondary lymphatic follicles (sLFs) were concentrated in the LP and submucosa  In LMFR, the localization of FcRn was similar to that in RMFR  InFR Figs.  and 8.FiThe localization of FcRn was in the two groups were both similar to those in young group.The localization of FcRn was in RMFR and LMFR were similar. It was highly expressed in macrophages and DCs in sLFs, however, the expression levels were very low in other tissues.1') In RMFR, the FcRn expression level in non-FAE was significantly higher than that in FAE in young, pubertal and middle-aged groups, respectively (P < 0.05)   In LMFR, the FcRn expression level in non-FAE was significantly higher than that in FAE in pubertal and middle-aged groups, respectively (P < 0.05)   Fig. , c and d05) Fig.  and e; (05) Fig.  and d, a05) Fig. , b and e(1') In RMFR, the FcRn expression level in FEA and non-FAE both gradually increased from fetus to pubertal groups with increasing age, peaked in pubertal group, and subsequently gradually declined  In LMFR, the FcRn expression level in FAE peaked in young and kept the high level to pubertal period, then subsequently significantly declined, but that in non-FAE gradually increased with increasing age, peaked in the middle-aged group   Fig. .Fig. 10COrganized MALT is a critical part of the mucosal immune system . Wang etP > 0.05), while that in FAE was significantly lower than that in non-FAE in pubertal and middle-aged groups, respectively (P < 0.05). The expression characteristics were similar to those of pIgR, transport receptor of SIgA, in this area [Compared with non-FAE, the FAE is a kind of specialized epithelium, on which the unique microfolds cell (M cell) could efficiently uptake and transport macromolecules and microorganisms in gut lumen to the underlying lymphoid tissue . The MODhis area . In ALNAIn addition, our results showed that FcRn was expressed in the vessel endothelium and smooth muscle in this area, which was similar to the FcRn expression characteristics in the vessel and smooth of mice and humans . It suggIn the present study, both macrophages and DCs in sLFs of ALNA in abomasum of Bactrian camels highly expressed FcRn, respectively. Researches have shown that there was mononuclear phagocyte system composed of monocytes, DCs and macrophages in sLFs. And in this system, different types of cells had different subtypes, respectively, and were distributed in special regions –53. TheyOur results showed that FcRn was mainly expressed in non-FAE, the effector sites, although which was expressed in FAE, the inductive sites for mucosal immunity. And it was also expressed in DCs and macrophages in sLFs of all ages of Bactrian camels. This provided a powerful evidence that IgG could participate in mucosal immune and immune response and tolerance in this area.


Next-generation sequencing (NGS) has opened up the possibility of detecting new viruses in unresolved diseases. Recently, astrovirus brain infections have been identified in neurologically diseased humans and animals by NGS, among them bovine astrovirus (BoAstV) CH13/NeuroS1, which has been found in brain tissues of cattle with non-suppurative encephalitis. Only a few studies are available on neurotropic astroviruses and a causal relationship between BoAstV CH13/NeuroS1 infections and neurological disease has been postulated, but remains unproven. Aiming at making a step forward towards assessing the causality, we collected brain samples of 97 cases of cattle diagnosed with unresolved non-suppurative encephalitis, and analyzed them by in situ hybridization and immunohistochemistry, to determine the frequency and neuropathological distribution of the BoAstV CH13/NeuroS1 and its topographical correlation to the pathology. We detected BoAstV CH13/NeuroS1 RNA or proteins in neurons throughout all parts of the central nervous system (CNS) in 34% of all cases, but none were detected in cattle of the control group. In general, brain lesions had a high correlation with the presence of the virus. These findings show that a substantial proportion of cattle with non-suppurative encephalitis are infected with BoAstV CH13/NeuroS1 and further substantiate the causal relationship between neurological disease and astrovirus infections. The first interest in viral infections of the central nervous system (CNS) dates back to antiquity , but neuAstroviruses are small, non-enveloped, single-stranded RNA viruses with a star-like shape when viewed in the electron microscope. Since the discovery of the first astrovirus in a human stool sample in 1975 , astroviUsing NGS, BoAstV has been found in the brains of cattle with non-suppurative encephalitis, termed BoAstV CH13 or BoAstV NeuroS1 ,7. As boThe postulates of Koch are still considered the gold standard to prove causal relationships of microbes and diseases. These postulates define the following requirements for proof of causation: (i) microorganisms are associated with a disease and its lesions, (ii) microorganisms can be isolated from the host and purely cultured, and (iii) the disease is reproducible by inoculation of healthy hosts and that the same microorganism can then be isolated from that host . While tFor BoAstV CH13/NeuroS1, the postulates of Koch have not yet been fulfilled, because the virus cannot be isolated in cell culture and a reverse genetics system is not yet available. In the present study, we aimed at taking the first step towards assessing causality, by following the suggestions of Lipkin and Anthony. We performed a large-scale retrospective investigation of unresolved cases of non-suppurative encephalitis in order to assess the statistical association of BoAstV CH13/NeuroS1 infection and neurological disease and its neuroanatomical correlation to the pathological lesions in cattle. The results provide important insights on the frequency of BoAstV CH13/NeuroS1 infection in the Swiss cattle population and further support it being a major cause of encephalitis in cattle.n = 52), and of animals with neurological disorders but with lesions indicating a bacterial origin (n = 6), under the same conditions. All cases were submitted to the neuropathological diagnostic services between 1985 and 2015. Tissue samples included formalin-fixed paraffin-embedded (FFPE) tissue of the CNS, mainly the brainstem, cerebellum, cerebrum and hippocampus. All tissues were analyzed by in situ hybridization (ISH) for the presence of BoAstV CH13/NeuroS1 RNA, and positive results were confirmed by immunohistochemistry (IHC). Tissues of cases with a known BoAstV CH13/NeuroS1 positive and negative status served as positive and negative controls.A total of 97 cases of histologically-confirmed bovine non-suppurative encephalitis of unresolved etiology were collected from the archives of the Vetsuisse Faculty in Bern and Zurich, Switzerland. All animals diagnosed were presenting with neurological symptoms, mainly gait abnormalities and behavioral changes. As negative controls, we analyzed samples of different brain regions of healthy animals without brain lesions  in Dako Target Retrieval solution, pH 9  as antigen retrieval method. Blocking was performed with 10% normal goat serum in phosphate-buffered saline (PBS)-Tween solution, after which the ORF2-con immunoserum (1:100) was added overnight at 4 °C. Antibody binding was detected by the Dako REAL Detection System according to the instructions of the manufacturer (Agilent Technologies). IHC and ISH grading was performed according to the number of stained neurons. Serial sections of all slides were submitted to hematoxylin-eosin staining for histopathological assessment of lesions.IHC was performed according to the validated ORF2-con protocol  and eachISH and IHC results were assessed using a semi-quantitative grading: (−) = no positive cells; (+) = 1–10 positive cells; (++) = 10–50 positive cells, and (+++) = >50 positive cells. Histopathological lesions were similarly graded as on haematoxylin and eosin (HE) stained slides: (−) = no lesions, (+) = mild lesions, (++) = moderate lesions, and (+++) = severe lesions. The assessment of both the ISH/ IHC and the histopathological grading was not performed on specific microscopic fields but on complete sections. The topographical correlation of lesions with BoAstV CH13/NeuroS1 detection was judged as good correlation, partial correlation, and the absence of correlation. A good correlation is seen in sections where the virus is located in the same microscopic field, affecting the same neuroanatomical area that shows the highest grade of lesions. A partial correlation does show an overlap in the presence of lesions and the virus, while not precisely the same, but a close-by neuroanatomical area is affected, or when major lesions comprise glial nodes or perivascular cuffs, but not neurons. Absence of correlation reflects a very large discrepancy between lesions and the presence of viral RNA (vRNA) or proteins .Statistical analysis was done by Fisher’s exact test using the Prism software .The ISH results of all available brain regions of 97 cases of bovine non-suppurative encephalitis revealed that 33 cases were positive for BoAstV CH13/NeuroS1 RNA (34%) by both ISH probes and in at least one region of the CNS, while none of the 58 negative controls showed reactivity, neither with ISH-probe A, nor with probe B, under the same conditions . In BoAsWe analyzed all 33 cases that were BoAstV CH13/NeuroS1-positive in the ISH by IHC to confirm the results. For this we chose the sections with the strongest positive ISH signal from each case and compared the IHC result directly to the ISH result. In 27 BoAstV CH13/NeuroS1 positive cases the staining in the IHC was comparable to the one in the ISH, not only in the region but also in the number of cells affected by the virus. It appears that approximately the same number of neurons showed a labeling in the same neuroanatomical region by both methods. However, in six astrovirus-positive cases we could not detect an ORF2-con specific signal in the IHC despite a strong neuronal staining of a serial section by ISH. Based on repetitively positive signals by both ISH-probes in at least two brain regions, we still considered these cases as BoAstV CH13/NeuroS1-positive.p < 0.0001).Taken together, the BoAstV CH13/NeuroS1 infection that we detected by ISH and IHC in approximately 1/3 of cattle diagnosed with non-suppurative encephalitis, while negative controls remained unstained, clearly shows a correlation with the disease, which is supported by an extremely highly significant statistical correlation , pathologies of the gastrointestinal tract , and nystagmus. On the other hand, astrovirus-negative non-suppurative cases had slightly higher instances of gait abnormalities, opisthotonus, ataxia, fever, and recumbency .n = 10) in which a whole set of representative brain regions was available by ISH and IHC on serial sections , which are frequently used for human enteric astroviruses , and in Studies aiming at understanding other possible viruses causing bovine non-suppurative encephalitis, at developing in vitro systems, as well as tools for serological diagnostics and research into disease epidemiology, should be encouraged to further expand our understanding of the causes and significance of neuroinfectious diseases, which potentially threaten animal and public health.


MGMT, 1p19q, and ATRX; being perceived as most problematic. Neuropathologists advocate the need for international harmonization of standards and consensus guidelines, and the majority is willing to actively engage in interlaboratory trials aiming at quality control  in order to assess 1) the extent to which molecular markers have already been incorporated in glioma diagnoses, 2) which molecular techniques are in daily use, and 3) to set a baseline for future surveys in this field. Based on 130 responses from participants across 40 nations neuropathologists uniformly rate molecular marker testing as highly relevant and already incorporate molecular information in their diagnostic assessments. At the same time however, the survey documents substantial differences in access to crucial biomarkers and molecular techniques across geographic regions and within individual countries. Concerns are raised concerning the validity of test assays with  control .  The 2016 update of the WHO classification of tumors of the central nervous system has introduced an integrated diagnostic approach that incorporates both tumor morphology and molecular information [IDH mutations, 1p19q codeletion, and ATRX mutations are meanwhile considered prerequisites for precise subtyping into diffuse astrocytoma, oligodendroglioma, and glioblastoma categories [TERT promoter mutations have been implicated as relevant prognostic factors [MGMT promoter methylation status further impacts therapeutic choices in elderly patients [IDH1 R132H mutation and ATRX mutation with subsequent loss of ATRX protein expression, are mainly evaluated by immunohistochemistry [IDH2, rare IDH1, and TERT mutations, as well as fluorescence in situ hybridization (FISH) or whole chromosomal arm spanning multiplex ligation-dependent probe amplification (MLPA) for 1p19q codeletion testing [MGMT promoter methylation status is commonly evaluated using pyrosequencing or methylation-specific polymerase chain reaction (MSP) [Adult diffuse glioma comprises the most common group of primary brain tumors and thus, represents a major workload for surgical neuropathologists. It is a prototypic example of a group of tumors that underwent substantial regrouping based on molecular constellations . Molecultegories , 4, 5. Lpatients . All thehemistry . All oth testing . Likewison (MSP) . More adon (MSP) , 12. We herein sought to document current practice patterns in the molecular assessment of adult diffuse glioma during a period of transition after the revised WHO classification was released earlier in 2016. We specifically sought to: 1) assess the extent to which the different molecular markers have already been incorporated into routine practice, 2) assess which molecular techniques and platforms are in daily use or will be implemented in the near future, and 3) to set a baseline evaluation for future surveys in this area. th European Congress of Neuropathology (ECNP) in Bordeaux, France, July 6 – 9, 2016. In addition to the congress participants (294 registrations), all Euro-CNS members were invited via email to complete the survey online. It was designed and tested to allow a completion time of ~ 5 minutes. Responses were anonymously collected and stored online using Google spreadsheet. The hyperlink to the online survey was accessible through October 17, 2016. Summary statistics were prepared for responses to each question and presented as percentage or absolute number of responses. Test statistics included χ2-test for categorical variables and t-test for numeric values. All statistical analyses and graphic representations were performed with Microsoft Excel v14.4.8, SPSS-statistics v23, and MATLAB R2015b. The World 2016 ranking of gross domestic products (GDP) per capita was used to stratify countries into high- (top 20% GDP per capita) vs. lower-income countries (lower 80% GDP per capita). Subgroup analysis was performed for countries with more than 10 respondents, respectively (top-responding countries). Detailed information on the number of practicing neuropathologists actively engaged in brain tumor diagnostics per country was not available. The instrument of choice for this study was a survey targeting neuropathologists engaged in brain tumor diagnostics using the European Confederation of Neuropathological Societies (Euro-CNS) as communication platform. Euro-CNS aims at promoting and maintaining the harmonization of neuropathological training and practice across Europe (http://www.euro-cns.org). It comprises 21 national organizations with over 1,000 individual memberships. The survey included questions pertaining to the overall significance of molecular marker testing, its impact on diagnostic evaluations, current and near future availability of molecular platforms, and relevance of quality control such as ring trials and consensus guidelines. Participants were asked to provide information on current place of work. Most questions offered multiple choices with a broad range of answers presented in random order. The survey was designed by the investigators and reviewed by members of the Euro-CNS executive team prior to its release in paper- and web-based format on occasion of the 11A total of 130 participants completed the survey. 45 responses were collected at the occasion of ECNP 2016 and another 85 respondents followed the invitation by email. The vast majority of respondents  indicated neuropathology or general pathology with sub-specialization in neuropathology as their profession. In contrast, respondents from other disciplines were rare and included molecular biologists (4 respondents), neurologists (3 respondents), or neurosurgeons (1 respondent). Overall, responses were collected from 40 different countries across all geographic regions with 75% of respondents indicating to work in Europe followed by North America (11%) and Asia (9%) . Thus, 7IDH1 (93.1%), 1p19q (85.4%), and MGMT (70.0%), whereas TERT (17.7%) is the least widely evaluated marker . However, substantial within-country heterogeneity in the number and combination of markers is observed when restricting the analysis to top-responding countries only, which collectively fall in the high-income category . The overwhelming majority of neuropathologists considers molecular testing as important or very important (93.6%) and already incorporates molecular information to some extent in their routine diagnostic assessments (96.0%) . Among td marker . 64% of category . 50% of category . Those aMGMT (20 nominations), 1p19q (16 nominations), and ATRX (15 nominations); they considered most problematic (FISH is by far the most broadly available technique (71.5%), followed by gene panel sequencing (46.2%), and MSP (45.4%) . In contblematic . Concernblematic . The present Euro-CNS survey documents practice patterns pertaining to the molecular classification of adult diffuse glioma. It stands as one of the first efforts that specifically focus on neuropathologists who are responsible for implementing the new “integrated” diagnostic format as advocated by the updated WHO classification. Even though our survey has a strong focus on the European neuropathology community and thus might not be fully representative for the international scenario, it provides useful insights and complements previous surveys in this field , 14. Overwhelmingly positive responses underscore the broad support among neuropathologists towards the integration of molecular markers in their daily routine. This positive attitude is in line with a previous questionnaire among members of the Society of Neuro-Oncology . In theiIDH1, 1p19q, and MGMT are probably the best-established ones and their diagnostic, prognostic, and predictive relevance has been demonstrated and confirmed in several independent studies [MGMT being the least available. At the same time, MGMT testing is perceived most problematic, which points towards substantial technical issues that prevent its smooth translation into routine use. Similar reasons might account for the less prevalent use of IDH2 testing, which – in contrast to the most common IDH1 R132H mutation – definitely requires a sequencing-based approach. Among the various markers, TERT mutation is the most recently implicated one and not explicitly included in the WHO classification [The present survey focused on a set of molecular markers that are meanwhile considered crucial in the assessment of adult diffuse glioma. Among the proposed markers,  studies , 16, 17.fication . Thus, iThe main purpose of the WHO tumor classification is to provide a consensus classification for measuring cancer burden in a standardized way worldwide. It is not the task of WHO to provide guidelines on which molecular tests to use. Accordingly, the updated WHO brain tumor classification provides no recommendations pertaining to which molecular techniques to choose. As recognized international or worldwide guidelines do not exist up to now, there is ample freedom left to the individual centers. This may pose certain problems as different techniques are associated with inherent advantages and disadvantages and thus, leave room for inter-laboratory differences. The most widely available molecular technique and seemingly standard in (neuro)pathology laboratories is FISH, a cost-effective but laborious and time-intense method that offers the advantage of morphology-based evaluations, which per se are intuitive to pathologists. On the other end of the spectrum is array-based DNA methylation profiling, which is available to only very few neuropathologists mostly based in Europe and the United States . ®  [Overall, more than half of all neuropathologists report the routine use of 3 or more molecular platforms. The combination of techniques, however, varies substantially without emerging “optimal” technical set-up. Hence, some observations deserve to be mentioned. 1) A substantial number of neuropathologists who routinely use panel-sequencing report to no longer perform targeted gene sequencing. 2) Similar mutual trends are observed for MSP vs. pyro-sequencing and FISH vs. MLPA with clear preferences for MSP and FISH, respectively. 3) Most importantly, while some institutions have access to almost all platforms, a considerable number of neuropathologists report to have no or only limited access to molecular techniques, thus, indicating substantial inequalities of diagnostic standards across geographic regions and institutions. This is well in line with a previous survey of the International Society of Neuropathology that demonstrated huge differences in access to molecular techniques – not only on a global scale but also within Europe . These dMA, USA)  or IBM WIDH mutations, methylation status of the MGMT gene promoter, and copy numbers of chromosomal arms 1p19q) and 2) makes use of epigenetic signatures as tracers for tissues/cells of origin, which might aid in diagnostically challenging cases [Over the last years, DNA methylation profiling has emerged as a particularly appealing candidate technique for brain tumor diagnostics as it 1) allows for the simultaneous assessment of multiple markers  Many neuropathologists have a different focus apart from brain tumors such as neurodegenerative or neuromuscular diseases. 2) Neuropathologists with limited available resources and frank problems to implement new markers might be less motivated to participate in a targeted survey. 3) Ultimately, it is difficult to estimate the maximum feasible target population, i.e., currently practicing neuropathologists per country as Euro-CNS members comprise various disciplines including basic neuroscientists and related clinical disciplines. The survey was designed to provide summary views on molecular markers and access to molecular techniques, rather than enabling detailed linkage of the individual markers with their corresponding techniques. That, however, would be of interest and future surveys would benefit from including this information. Similarly, it would be reasonable to extend this approach to other brain tumor entities and additional markers. MGMT, 1p19q, and ATRX being perceived as most problematic. Uniformly, neuropathologists advocate the need for international consensus guidelines and the majority is willing to actively engage in quality control, harmonization of standards, and interlaboratory comparisons by means of ring trials. In summary, the current Euro-CNS survey demonstrates broad support of the neuropathology community towards the integration of molecular markers in routine diagnostic assessments as advocated by the updated WHO classification. Despite enthusiasm, access to molecular markers and techniques varies substantially across geographic regions and within individual countries. In addition, major concerns are raised pertaining to the validity of test assays with We appreciate the time investment and active participation of all survey respondents. We further thank the Euro CNS executive team and ECNP 2016 congress officials for providing the framework and Ilja Huang, Euro-CNS Administrator, for excellent technical support. We acknowledge Dr. Bernhard Baumann, Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Austria, for assistance with graphic data representation and Dr. David N. Louis, Chair of Massachusetts General Pathology and Harvard Medical School, for constructive discussion and valuable feedback. The authors declare no conflict of interest.


VTA) regulate SFA and AHP (together referred to as intrinsic inhibition) in the prefrontal cortex (PFC) remains unclear. Using whole cell electrophysiology, we measured intrinsic inhibition in prelimbic (PL) layer 5 pyramidal cells of male adult rats. Results demonstrate that bath application of dopamine reduced intrinsic inhibition (EC50: 25.0 μmol/L). This dopamine action was facilitated by coapplication of cocaine (1 μmol/L), a blocker of dopamine reuptake. To evaluate VTA dopamine terminals in PFC slices, we transfected VTA dopamine cells of TH::Cre rats in vivo with Cre‐dependent AAVs to express channelrhodopsin‐2 (ChR2) or designer receptors exclusively activated by designer drugs (DREADDS). In PFC slices from these animals, stimulation of VTA terminals with either blue light to activate ChR2 or bath application of clozapine‐N‐oxide (CNO) to activate Gq‐DREADDs produced a similar reduction in intrinsic inhibition in PL neurons. Electrophysiological recordings from cells expressing retrograde fluorescent tracers showed that this plasticity occurs in PL neurons projecting to the accumbens core. Collectively, these data highlight an ability of VTA terminals to gate intrinsic inhibition in the PFC, and under appropriate circumstances, enhance PL neuronal firing. These cellular actions of dopamine may be important for dopamine‐dependent behaviors involving cocaine and cue‐reward associations within cortical–striatal circuits.Spike frequency adaptation (SFA or accommodation) and calcium‐activated potassium channels that underlie after‐hyperpolarization potentials (AHP) regulate repetitive firing of neurons. Precisely how neuromodulators such as dopamine from the ventral tegmental area ( Reward learning involves burst firing of midbrain dopamine neurons in the ventral tegmental area (VTA) Schultz, , 2002. TThe prelimbic (PL) area of the rodent medial PFC is anatomically and functionally distinct  function to reduce repetitive firing, allowing neurons to adapt to constant, low‐intensity excitation, and yet still respond to additional brief, intense depolarizing inputs , plays an important role in neuronal plasticity and learning  were used following acclimation to the vivarium (>7 days). Rats were single or double housed in a temperature‐ and humidity‐controlled vivarium under a reverse 12:12‐h light/dark cycle with free access to food and water. Prior to surgical procedures, which occurred in the light in a separate surgical suite, rats were anesthetized with a ketamine HCl/xylazine mixture  followed by ketorolac  and cefazolin .All procedures were in accordance with the MUSC Institutional Animal Care and Use Committee. Adult (>P55) male Sprague Dawley (Charles River) and TH::Cre+ transgenic rats (P56–70) received bilateral injections of AAV2  with the following constructs from the University of North Carolina Vector Core: EF1a‐DIO‐hChR2(H134R)‐EYFP (n = 6), hSyn‐DIO‐rM3D(Gs)‐mCherry (n = 4), or hSyn‐DIO‐hM3D(Gq)‐mCherry (n = 10) into VTA: AP −5.5 mm, ML ±0.8 mm, DV −8.15 mm (with respect to bregma). Four rats also received intracranial injections of fluorescent microspheres  into the NAcC . From four microsphere injected rats, locations into the NAcC were confirmed in acute brain slices fixed with 4% paraformaldehyde; one rat was excluded for missed placement. All injections were made through a glass micropipette (30–40 μm diameter tip) using a Nanoinject II (Drummond Scientific). Pipettes remained in place for at least 5 min to minimize diffusion along the pipette track. Following 4–9 weeks of incubation, rats were sacrificed for immunohistochemistry or electrophysiology experiments.μm. ChR2 and hM3D(Gq) receptor expression was visualized with immunohistochemistry for EYFP or mCherry tags. Dopamine cells were identified as tyrosine hydroxylase (TH) immunoreactive  vector in TH::Cre rats  containing the PFC (+3.0–3.7 AP from bregma). Slices were incubated (32°C) in aCSF with 0.01 mmol/L MK‐801 for at least 30 min prior to experimentation. Whole cell patch clamp experiments were performed on large pyramidal shaped cells in layer 5 prelimbic cortex, visualized using an Olympus BX51WI (Olympus America) equipped with gradient contrast infrared optics. Neurons expressing fluorescent microspheres were detected with collimated LED light  emitted through a 60X water immersion lens. The aCSF recording solution was identical to the incubation aCSF, except MK‐801 was excluded. Brain slices were perfused using a gravity‐fed system perfused at a flow rate of 2–3 mL per min and maintained at physiological temperature (~32°C). Recordings were made with MultiClamp700B (Molecular Devices). Patch electrodes (2–5 MΩ) were filled with internal solution containing 115 mmol/L K‐methylsulfate, 20 mmol/L NaCl, 1.5 mmol/L MgCl2, 10 mmol/L HEPES, 0.1 mmol/L EGTA, 2 mmol/L ATP sodium salt hydrate, and 0.3 mmol/L GTP sodium salt hydrate . Membrane potentials for these pyramidal neurons typically ranged between −65 and −75 mV, with spike potentials above 0 mV, consistent with previous reports  containing 126 mmol/L NaCl, 2.5 mmol/L KCl, 1.2 mmol/L MgClSFA was measured in current clamp configuration from a holding potential of −70 mV with input–output (I‐O) curves similar to previous reports   with support by NIH‐NCI grant number X01 NS064882‐0. Cocaine was provided by NIDA Drug Supply Program. Dopamine was purchased from Sigma. All other compounds were purchased from Abcam/Ascent Scientific.ChR2 stimulation occurred with purpose built collimated LED light  emitted through a 60X water immersion lens. For measurements of action potential firing during ChR2 photostimulation, VTA terminals were stimulated with blue light (473 nm). Each photostimulation episode consisted of eight trains of 10 msec light pulses applied at 17 Hz. This strategy was designed to mimic phasic dopamine neuron firing  versus response curves were used to fit dopamine dose–response curves, with EC50 values reported. For analysis of within cells correlation between our two metrics of intrinsic inhibition, AHP and action potential firing datasets were fit by linear regression, followed by a Pearson's correlation. Multiple comparison posttests were used as indicated. Ps < 0.05 were considered significant.All data (mean ± SEM) were analyzed in Axograph, Excel, and Prism. For comparisons of pre‐ and postdrug input–output curves within cells, all drugs compounds remained in the aCSF during postdrug measurements. Data were analyzed with two‐way ANOVAs matching both factors (cell and current injection), or paired and unpaired μmol/L) reduced intrinsic inhibition when spiking was triggered by current pulses of 1000 pA  induced a significant shift in spike frequency at current injections at 0.8 and 1 nA, with a trend toward a reduction at current injections between 0.4 and 0.8 nA , a blocker of dopamine reuptake  layer 5 pyramidal cells identified by location, morphology, and electrophysiological criteria . We assayed intrinsic inhibition using two interrelated metrics: SFA and AHP. SFA of action potential firing was measured in current clamp mode in response to depolarizing injections of current (0.1–1 nA steps of 800 msec)  promoter (TH::Cre rats) ‐EYFP (“AAV‐ChR2”) bilaterally into the VTA .For electrophysiological experiments, the remaining five AAV‐injected TH::Cre+ rats with AAV‐hSyn‐DIO‐hM3D(Gq)‐mCherry and 4 TH::Cre+ rats with AAV‐hSyn‐DIO‐hM3D(Gs)‐mCherry , similar to previous reports . For electrophysiological experiments, acute PFC brain slices were prepared from the remaining DREADD‐injected rats (4–9 weeks after injection) to record pyramidal cells in L5 PL‐PFC  to stimulate Gq‐DREADD‐expressing VTA terminals reduced SFA at current injections above 0.4 nA . We noted similar, but less robust effects with CNO stimulation of Gs‐DREADD expressing VTAPL fibers in that SFA, but not the AHP, was significantly reduced by CNO (Gs‐DREADDs—APs: t(5) = 2.846, P = 0.036 at 1 nA of injected current; AHP: t(5) = 0.42, P = 0.6907). Together, these results indicate stimulation of VTAPL terminals with DREADDs reduces intrinsic inhibition of pyramidal cells in the PL.We evaluated the effects of stimulating VTAPFC Fig. C. As exp nA Fig. D. The in nA Fig. E and the nA Fig. F in respPL fibers altered intrinsic inhibition in PL cells innervating the NAcC. We again transfected the VTA of TH::Cre+ transgenic rats with Gq‐DREADDs, and also injected fluorescently labeled beads in NAcC to retrogradely label PL neurons in this circuit (Fig. PL fibers with CNO significantly reduced SFA at current injections above 0.5 nA (Fig. PL terminals shape intrinsic inhibition in PFC cells projecting to the NAcC, it remains to be determined whether this is a circuit‐specific action, or whether other circuits, for example, PFC cells projecting to the nucleus accumbens shell, show similar sensitivity to VTA terminal stimulation.We next determined if chemogenetic stimulation of VTAuit Fig. G. We maduit Fig. H. We obs nA Fig. I and the nA Fig. J. While PL terminals reduces intrinsic inhibition in PL cells that innervate the NAcC, a circuit known to be important for a variety of dopamine‐dependent behaviors.Collectively, these data suggest that activation of VTAThe prefrontal cortex is important for behavioral flexibility, working memory, and cognitive control. The VTA, in general, and dopamine, in particular, have an essential role in these processes (Naneix et al., The most important aspect of this study was the experiments showing a slow, progressive change in SFA and the AHP in PFC cells during photoactivation or chemogenetic stimulation of VTA axon terminals. Neither blue light nor CNO produced any changes in tissue from animals not transfected with virus. Yet, SFA/AHP declined reliably during the exposure to repeated phasic‐like photoactivation patterns or bath application of dopamine with cocaine, a blocker of dopamine reuptake, or CNO activation of Gq‐DREADDs. A logical interpretation of these data is that the phasic stimulation of VTA axons and blockade of dopamine reuptake or the continuous stimulation of axons with DREADDs caused a buildup of extracellular dopamine in the slice that activated dopamine receptors. This scenario would parallel results from another optogenetic study in the nucleus accumbens showing stimulation of dopamine release is both possible and efficient with similar low‐light power levels (Lu et al., An alternative scenario is that photo/chemostimulation of these terminals released glutamate, which increased spiking. Prior studies show a population of VTA dopamine neurons that also express vesicular glutamate transporter 2 make glutamatergic connections with regions such as the nucleus accumbens (Mingote et al., Instead, our study examined the response to bath applied dopamine, and observed similar robust changes in SFA/AHP that showed increased sensitivity during bath application of cocaine. Although we did not determine the receptor subtype activated by dopamine, numerous previous studies have addressed the relevant cellular actions of dopamine D1‐ and D2‐like receptors that orchestrate an “inverted‐U” PFC response to dopamine (Trantham‐Davidson et al., Other studies at the systems level support the functional association between D1 receptors in the PFC, dopamine terminals emanating from the VTA, and PFC projections to the nucleus accumbens. Optogenetic activation of VTA dopamine cells facilitates temporal control for some tasks via recruitment of D1‐expressing PFC cells (Narayanan et al., These results suggest that dopamine release from VTA terminals gates intrinsic inhibition in the cortex by regulating SFA and the AHP. This plasticity was induced with burst‐like stimulation of VTA terminals, in cortical neurons that project to the accumbens core. Dopamine‐induced reductions in intrinsic inhibition were potentiated by blockade of dopamine reuptake with acute exposure to cocaine. Such a mechanism may contribute to the VTA regulation of PL‐PFC activity during the presentation of rewards and their cues.None declared.