Automatic quantification of lymphocyte vacuolization in peripheral blood smears of patients with Batten's disease (CLN3 disease).

Quantifying lymphocyte vacuolization in peripheral blood smears (PBSs) serves as a measure for disease severity in CLN3 disease-a lysosomal storage disorder of childhood-onset. However, thus far quantification methods are based on labor-intensive manual assessment of PBSs. As machine learning techniques like convolutional neural networks (CNNs) have been deployed quite successfully in detecting pathological features in PBSs, we explored whether these techniques could be utilized to automate quantification of lymphocyte vacuolization. Here, we present and validate a deep learning pipeline that automates quantification of lymphocyte vacuolization. By using two CNNs in succession, trained for cytoplasm-segmentation and vacuolization-detection, respectively, we obtained an excellent correlation with manual quantification of lymphocyte vacuolization (r = 0.98, n = 40). These results show that CNNs can be utilized to automate the otherwise cumbersome task of manually quantifying lymphocyte vacuolization, thereby aiding prompt clinical decisions in relation to CLN3 disease, and potentially beyond.

Quantifying lymphocyte vacuolization serves as a measure of CLN3 disease severity.

BACKGROUND: The CLN3 disease spectrum ranges from a childhood-onset neurodegenerative disorder to a retina-only disease. Given the lack of metabolic disease severity markers, it may be difficult to provide adequate counseling, particularly when novel genetic variants are identified. In this study, we assessed whether lymphocyte vacuolization, a well-known yet poorly explored characteristic of CLN3 disease, could serve as a measure of disease severity. METHODS: Peripheral blood obtained from healthy controls and CLN3 disease patients was used to assess lymphocyte vacuolization by (a) calculating the degree of vacuolization using light microscopy and (b) quantifying expression of lysosomal-associated membrane protein 1 (LAMP-1), using flow cytometry in lymphocyte subsets as well as a qualitative analysis using electron microscopy and ImageStream analysis. RESULTS: Quantifying lymphocyte vacuolization allowed to differentiate between CLN3 disease phenotypes (P = .0001). On immunofluorescence, classical CLN3 disease lymphocytes exhibited abundant vacuole-shaped LAMP-1 expression, suggesting the use of LAMP-1 as a proxy for lymphocyte vacuolization. Using flow cytometry in lymphocyte subsets, quantifying intracellular LAMP-1 expression additionally allowed to differentiate between infection and storage and to differentiate between CLN3 phenotypes even more in-depth revealing that intracellular LAMP-1 expression was most pronounced in T-cells of classical-protracted CLN3 disease while it was most pronounced in B-cells of "retina-only" CLN3 disease. CONCLUSION: Lymphocyte vacuolization serves as a proxy for CLN3 disease severity. Quantifying vacuolization may help interpretation of novel genetic variants and provide an individualized readout for upcoming therapies.

Improving home haemodialysis: Stability evaluation of routine clinical chemistry analytes in blood samples of haemodialysis patients.

INTRODUCTION: A growing number of dialysis patients is treated with home haemodialysis. Our current pre-analytical protocols require patients to centrifuge the blood sample and transfer the plasma into a new tube at home. This procedure is prone to errors and precludes accurate bicarbonate measurement, required for determining dialysate bicarbonate concentration and maintaining acid-base status. We therefore evaluated whether cooled overnight storage of gel separated plasma is an acceptable alternative. MATERIALS AND METHODS: Venous blood of 34 haemodialysis patients was collected in 2 lithium heparin blood collection tubes with gel separator (LH PSTTM II, REF 367374; Becton Dickinson, New Jersey, USA). One tube was analysed directly for measurement of bicarbonate, potassium, calcium, phosphate, glucose, urea, lactate, aspartate aminotransferase (AST), and lactate dehydrogenase (LD); whereas the other was centrifuged and stored unopened at 4 °C and analysed 24 h later. To measure analyte stability after 24 h of storage, the mean difference was calculated and compared to the total allowable error (TEa) which was used as acceptance limit. RESULTS: Potassium (Z = - 4.28, P < 0.001), phosphate (Z = - 3.26, P = 0.001), lactate (Z = - 5.11, P < 0.001) and AST (Z = - 2.71, P = 0.007) concentrations were higher, whereas glucose (Z = 4.00, P < 0.001) and LD (Z = 3.13, P = 0.002) showed a reduction. All mean differences were smaller than the TEa and thus not clinically relevant. Bicarbonate (Z = 0.69, P = 0.491), calcium (Z = - 0.23, P = 0.815) and urea (Z = 0.81, P =0.415) concentrations were stable. CONCLUSIONS: Our less complex, user-friendly pre-analytical procedure resulted in at least 24 h stability of analytes relevant for monitoring haemodialysis, including bicarbonate. This allows shipment and analysis the next day.

Temporally precise control of single-neuron spiking by juxtacellular nanostimulation.

Temporal patterns of action potentials influence a variety of activity-dependent intra- and intercellular processes and play an important role in theories of neural coding. Elucidating the mechanisms underlying these phenomena requires imposing spike trains with precisely defined patterns, but this has been challenging due to the limitations of existing stimulation techniques. Here we present a new nanostimulation method providing control over the action potential output of individual cortical neurons. Spikes are elicited through the juxtacellular application of short-duration fluctuating currents ("kurzpulses"), allowing for the sub-millisecond precise and reproducible induction of arbitrary patterns of action potentials at all physiologically relevant firing frequencies (<120 Hz), including minute-long spike trains recorded in freely moving animals. We systematically compared our method to whole cell current injection, as well as optogenetic stimulation, and show that nanostimulation performance compares favorably with these techniques. This new nanostimulation approach is easily applied, can be readily performed in awake behaving animals, and thus promises to be a powerful tool for systematic investigations into the temporal elements of neural codes, as well as the mechanisms underlying a wide variety of activity-dependent cellular processes.NEW & NOTEWORTHY Assessing the impact of temporal features of neuronal spike trains requires imposing arbitrary patterns of spiking on individual neurons during behavior, but this has been difficult to achieve due to limitations of existing stimulation methods. We present a technique that overcomes these limitations by using carefully designed short-duration fluctuating juxtacellular current injections, which allow for the precise and reliable evocation of arbitrary patterns of neuronal spikes in single neurons in vivo.

Genetic Mapping in Mice Reveals the Involvement of Pcdh9 in Long-Term Social and Object Recognition and Sensorimotor Development.

BACKGROUND: Quantitative genetic analysis of basic mouse behaviors is a powerful tool to identify novel genetic phenotypes contributing to neurobehavioral disorders. Here, we analyzed genetic contributions to single-trial, long-term social and nonsocial recognition and subsequently studied the functional impact of an identified candidate gene on behavioral development. METHODS: Genetic mapping of single-trial social recognition was performed in chromosome substitution strains, a sophisticated tool for detecting quantitative trait loci (QTL) of complex traits. Follow-up occurred by generating and testing knockout (KO) mice of a selected QTL candidate gene. Functional characterization of these mice was performed through behavioral and neurological assessments across developmental stages and analyses of gene expression and brain morphology. RESULTS: Chromosome substitution strain 14 mapping studies revealed an overlapping QTL related to long-term social and object recognition harboring Pcdh9, a cell-adhesion gene previously associated with autism spectrum disorder. Specific long-term social and object recognition deficits were confirmed in homozygous (KO) Pcdh9-deficient mice, while heterozygous mice only showed long-term social recognition impairment. The recognition deficits in KO mice were not associated with alterations in perception, multi-trial discrimination learning, sociability, behavioral flexibility, or fear memory. Rather, KO mice showed additional impairments in sensorimotor development reflected by early touch-evoked biting, rotarod performance, and sensory gating deficits. This profile emerged with structural changes in deep layers of sensory cortices, where Pcdh9 is selectively expressed. CONCLUSIONS: This behavior-to-gene study implicates Pcdh9 in cognitive functions required for long-term social and nonsocial recognition. This role is supported by the involvement of Pcdh9 in sensory cortex development and sensorimotor phenotypes.

Impaired fear extinction as displayed by serotonin transporter knockout rats housed in open cages is disrupted by IVC cage housing.

Anxiety disorders are influenced by both environmental and genetic factors. A well-known example for gene x environment interactions in psychiatry is the low activity (s) allelic variant of the serotonin transporter (5-HTT) promoter polymorphism (5-HTTLPR) that in the context of stress increases risk for depression and post-traumatic stress disorder (PTSD). Previously, we observed robust anxiety-related phenotypes, such as an impairment in fear extinction, in 5-HTT knockout (5-HTT-/-) versus wild-type (5-HTT+/+) rats housed in open cages. Recently, housing conditions were changed from open cages to individually ventilated cages (IVC), which are associated with a high ventilation fold and noise. This switch in housing conditions prompted an unplanned 5-HTT gene x environment interaction study in our rats. The current study shows that lifetime stress by means of IVC cage housing abolished genotype differences in fear extinction between 5-HTT-/- and 5-HTT+/+ rats. Although this effect was not attributed specifically to either the 5-HTT+/+ or the 5-HTT-/- genotype, the findings are in agreement with the modulatory role of serotonin in the processing of environmental stimuli. Our findings also underline the possibility that housing conditions confound the interpretation of anxiety-related behaviours in rodents.

The attribution of incentive salience to an appetitive conditioned cue is not affected by knockout of the serotonin transporter in rats.

Understanding the neurobiological basis underlying individual differences in conditioned stimulus (CS) sensitivity is pertinent, given that excessive conditioned responses to CSs is a key feature of anxiety-related disorders and drug addiction. We have previously shown that behaviour of serotonin transporter knockout (5-HTT(-/-)) rats-mimicking the common 5-HTT promoter polymorphism in humans-is strongly driven by Pavlovian CSs. To investigate whether the knockout rats attribute greater incentive salience to CSs, we tested the 5-HTT(-/-) rats and their wild-type counterparts in the sucrose-reinforced sign-versus goal-tracking task. We also assessed whether motivational properties of the unconditioned stimulus (sucrose pellet) are involved in the individual differences under investigation, by testing the animals in a sucrose-reinforced progressive ratio schedule of reinforcement. We found no genotype differences in sign-versus goal-tracking behavior, despite that progressive ratio responding was increased in 5-HTT(-/-) rats. In conclusion, the high CS sensitivity in 5-HTT(-/-) rats cannot be explained by enhanced incentive salience attribution to the CS as measured by the sign- versus goal-tracking paradigm. Rather, 5-HTT(-/-) rats may be more sensitive to the motivational properties of the unconditioned stimulus.

Perseverative instrumental and Pavlovian responding to conditioned stimuli in serotonin transporter knockout rats.

Environmental stimuli can influence behavior via the process of Pavlovian conditioning. Recent genetic research suggests that some individuals are more sensitive to environmental stimuli for behavioral guidance than others. One important mediator of this effect is serotonin transporter (5-HTT) genetic variance, which increases sensitivity to Pavlovian conditioned stimuli through changes in the build-up of corticolimbic circuits. As these stimuli can have reinforcing effects on instrumental responding, we here investigated their effects on instrumental behavior in 5-HTT knockout rats and their wild-type counterparts by means of the signal attenuation paradigm. In this paradigm animals acquired a Pavlovian association between a stimulus and food reward, and subsequently they had to lever press in order to gain access to this food reward-associated stimulus. Thereafter, half of the animals underwent extinction training during which extinction of the primary Pavlovian association was induced via non-reinforced stimulus presentations, whereas the other half did not receive this training. During a final test session all animals were tested for instrumental responding for the non-reinforced Pavlovian conditioned stimulus, as well as instrumental and Pavlovian responding to the stimulus after an initial lever-press. No genotype differences were observed during the training and extinction sessions. However, during the test session 5-HTT knockout rats that had not received prior extinction training displayed excessive instrumental responding. This was specifically observed during presentation of the stimulus (induced by the first lever press) and was accompanied by an increased number of feeder visits after termination of the stimulus presentation. An additionally performed c-Fos immunohistochemistry study revealed that the behaviors in these animals were associated with abnormal c-Fos immunoreactivity in the orbitofrontal cortex and basolateral amygdala, regions important for the acquisition and maintenance of Pavlovian conditioned stimuli. These findings complement earlier findings showing that 5-HTT knockout animals' behavior is heavily influenced by environmental stimuli and indicate that this extends to the instrumental domain.

Improved cognitive flexibility in serotonin transporter knockout rats is unchanged following chronic cocaine self-administration.

Cocaine dependence is associated with orbitofrontal cortex (OFC)-dependent cognitive inflexibility in both humans and laboratory animals. A critical question is whether cocaine self-administration affects pre-existing individual differences in cognitive flexibility. Serotonin transporter knockout (5-HTT(-/-) ) mice show improved cognitive flexibility in a visual reversal learning task, whereas 5-HTT(-/-) rats self-administer increased amounts of cocaine. Here we assessed: (1) whether 5-HTT(-/-) rats also show improved cognitive flexibility (next to mice); and (2) whether this is affected by cocaine self-administration, which is increased in these animals. Results confirmed that naïve 5-HTT(-/-) rats (n = 8) exhibit improved cognitive flexibility, as measured in a sucrose reinforced reversal learning task. A separate group of rats was subsequently trained to intravenously self-administer cocaine (0.5 mg/kg/infusion), and we observed that the 5-HTT(-/-) rats (n = 10) self-administered twice as much cocaine [632.7 mg/kg (±26.3)] compared with 5-HTT(+/+) rats (n = 6) [352.3 mg/kg (±62.0)] over 50 1-hour sessions. Five weeks into withdrawal the cocaine-exposed animals were tested in the sucrose-reinforced reversal learning paradigm. Interestingly, like the naïve 5-HTT(-/-) rats, the cocaine exposed 5-HTT(-/-) rats displayed improved cognitive flexibility. In conclusion, we show that improved reversal learning in 5-HTT(-/-) rats reflects a pre-existing trait that is preserved during cocaine-withdrawal. As 5-HTT(-/-) rodents model the low activity s-allele of the human serotonin transporter-linked polymorphic region, these findings may have heuristic value in the treatment of s-allele cocaine addicts.

Serotonin transporter knockout rats show improved strategy set-shifting and reduced latent inhibition.

Behavioral flexibility is a cognitive process depending on prefrontal areas allowing adaptive responses to environmental changes. Serotonin transporter knockout (5-HTT(-/-)) rodents show improved reversal learning in addition to orbitofrontal cortex changes. Another form of behavioral flexibility, extradimensional strategy set-shifting (EDSS), heavily depends on the medial prefrontal cortex. This region shows functional changes in 5-HTT(-/-) rodents as well. Here we subjected 5-HTT(-/-) rats and their wild-type counterparts to an EDSS paradigm and a supplementary latent inhibition task. Results indicate that 5-HTT(-/-) rats also show improved EDSS, and indicate that reduced latent inhibition may contribute as an underlying mechanism.

Behavioural therapy based on distraction alleviates impaired fear extinction in male serotonin transporter knockout rats.

BACKGROUND: The "biological susceptibility" model posits that some individuals, by genetic predisposition, are highly sensitive to environmental stimuli. Exposure to adverse stimuli leads to negative outcomes, and better outcomes follow favourable stimuli. Recent studies indicate that individuals carrying the low-activity (short; s) variant of the serotonin transporter (5-HTT)-linked polymorphic region (5-HTTLPR) show an enhanced vulnerability to posttraumatic stress disorder (PTSD). Simultaneously, they respond poorly to exposure therapy, the first-line treatment to enhance fear extinction in individuals with PTSD. Given that s-allele carriers also show improved adaptive responding when exposed to positive stimuli, we hypothesized that this trait could be used to offset impaired fear extinction. METHODS: We explored this hypothesis preclinically using wild-type and 5-HTT knockout (5-HTT-/-) male rats (n = 36) that share behavioural similarities with 5-HTTLPR s-allele carriers. Subsequent to cued fear conditioning, animals were tested for short- (1 and 2 days postconditioning) and long-term (6 days postconditioning) fear extinction in the absence or presence of a secondary "distracting" stimulus predicting the delivery of sucrose pellets. RESULTS: Introducing a secondary stimulus predicting sucrose pellets that distracts attention away from the fear-predicting stimulus led to a long-lasting improvement of impaired fear extinction in 5-HTT-/- male rats. LIMITATIONS: The contextdependency of the efficacy of the "distraction therapy" was not tested. In addition, it remains to be clarified whether the positive valence of the distracting stimulus is critical for the distraction of attention or whether a neutral and/or novel stimulus can induce similar effects. Finally, although of lesser importance from a therapeutic perspective, underlying mechanisms remain to be elucidated. CONCLUSION: These data indicate that positive environmental stimuli can be used to offset heightened responses to negative stimuli, particularly in individuals characterized by inherited 5-HTT downregulation and high sensitivity to environmental stimuli.

The interplay between brain 5-hydroxytryptamine levels and cocaine addiction.

Cocaine addiction is a major health problem that affects millions of people. Cocaine acts by inhibiting dopamine, noradrenaline and serotonin [5-hydroxytryptamine(5-HT)] reuptake. The dopaminergic system is generally assumed to be involved in the reinforcing aspects of the drug, but the role of 5-HT in the addictive potential of cocaine is unclear. In light of pharmacological manipulations and cocaine use-related disease states affecting brain 5-HT levels, we review studies on the effect of cocaine on central 5-HT function. In addition, the contribution of 5-HT to the rewarding, aversive, discriminative and subjective, as well as the motivational and reinforcing effects of cocaine is discussed. We specifically focus on net changes in the extracellular 5-HT levels that occur as a consequence of acute and chronic cocaine exposure and how these influence cocaine abuse-related behaviour. Overall, the data indicate that 5-HT plays a major role in the psychomotor stimulant, rewarding and discriminative stimulant effects of cocaine, but also affects the motivational and reinforcing effects of the drug. In addition, 5-HT mediates, to some extent, the aversive effects of cocaine. Difficulties with data interpretation are discussed.

Serotonin transporter genotype x construction stress interaction in rats.

A well-known example for gene x environment interactions in psychiatry is the one involving the low activity (s) allelic variant of the serotonin transporter (5-HTT) promoter polymorphism (5-HTTLPR) that in the context of stress increases risk for depression. In analogy, 5-HTT knockout rodents are highly responsive to early life, but also adult external stressors, albeit conflicting data have been obtained. In our study on emotion and cognition using homozygous 5-HTT knockout (5-HTT(-/-)) and wild-type (5-HTT(+/+)) rats we have been confronted with animal facility construction, which were associated with severe lifetime stress (noise and vibrations). To assess the impact of construction stress on well-established 5-HTT(-/-) rat phenotypes we conducted ad hoc analyses of 5-HTT(-/-) and 5-HTT(+/+) rats that grew up before and during the construction. The reproductive capacity of the parents of the experimental 5-HTT(+/-) rats was significantly decreased. Further, 5-HTT(-/-) anxiety-related phenotypes in the elevated plus maze and social interaction tests were abolished after construction noise exposure, due to increased anxiety in 5-HTT(+/+) rats and decreased anxiety in 5-HTT(-/-) rats (social interaction test only). In addition, reversal learning was improved in 5-HTT(+/+) and, to a milder extent, decreased in 5-HTT(-/-) rats. Finally, construction stress genotype-independently increased behavioural despair in the forced swim test. In conclusion, severe construction stress induces 5-HTT genotype-dependent 'for-better-and-for-worse' effects. These data importantly contribute to the understanding of 5-HTT gene x environment interactions and show the risk of losing genotype effects by construction stress.

Orbitofrontal cortex and amygdalar over-activity is associated with an inability to use the value of expected outcomes to guide behaviour in serotonin transporter knockout rats.

A disturbance in 5-HT signalling can lead to maladaptive and disruptive behavioural changes seen in neuropsychiatric disorders, potentially by 5-HT's role in cognitive control over behaviour. 5-HT levels are tightly controlled by the serotonin transporter (5-HTT). We and others have observed that 5-HTT availability affects reversal learning. Here we investigated the role of 5-HT in another type of cognitive control, which is the ability to use the value of expected outcomes to guide behaviour. 5-HTT knockout (5-HTT(-/-)) rats and wild-type (5-HTT(+/+)) controls were subjected to a Pavlovian reinforcer devaluation paradigm, which assesses the ability of an appetitive conditioned stimulus (CS) to gain access to the motivational properties of an upcoming aversive unconditioned stimulus (US). Neural correlates were evaluated using c-Fos immunohistochemistry, in brains of animals sacrificed 90min following the start of the probe test. Results show that conditioned responding was decreased in 5-HTT(+/+), but not 5-HTT(-/-), rats after US devaluation. In addition, OFC and basolateral amygdala (BLA) c-Fos immunoreactivity was increased in non-devalued 5-HTT(-/-) rats compared to non-devalued 5-HTT(+/+) rats. Whereas US devaluation increased c-Fos immunoreactivity in the OFC and BLA of 5-HTT(+/+) rats, there was no further increase in c-Fos immunoreactivity in the OFC and BLA of 5-HTT(-/-) rats. Taken together, 5-HTT(-/-) rats are unable to use the value of expected outcomes to guide behaviour, potentially due to over-activity of the OFC and BLA. Our findings suggest a new modulatory role of 5-HT in cognitive control over behaviour, which may have important implications for psychopathologies, like anxiety disorders and addiction.