Viloxazine Increases Extracellular Concentrations of Norepinephrine, Dopamine, and Serotonin in the Rat Prefrontal Cortex at Doses Relevant for the Treatment of Attention-Deficit/Hyperactivity Disorder.

Background: Viloxazine ER (viloxazine extended-release capsules; Qelbree®), a nonstimulant attention-deficit/hyperactivity disorder (ADHD) treatment, has known activity as a norepinephrine (NE) transporter (NET) inhibitor. In vitro studies have also shown direct pharmacological effects on specific serotonin (5-HT) receptors, but not on the serotonin transporter (SERT). An in vivo microdialysis study in rats showed viloxazine (50 mg/kg i.p.) increased extracellular 5-HT, NE, and dopamine (DA) in the prefrontal cortex (PFC), a key brain region in ADHD pathology. This study evaluated whether these effects occur at clinically relevant concentrations. Methods: Microdialysis experiments were conducted in freely-moving, Sprague-Dawley rats (males, 8 weeks). Viloxazine (1, 3, 10, 30 mg/kg) was administered intraperitoneally to establish the dose range in rats at which viloxazine plasma concentrations aligned with those of individuals with ADHD administered therapeutic doses of viloxazine ER. Concentrations of unbound viloxazine, NE, 5-HT, DA, and NE and 5-HT metabolites (3,5-dihydroxyphenylglycol [DHPG] and 5-hydroxyindoleacetic acid [5-HIAA]) were measured in PFC interstitial fluid. After identifying a therapeutically relevant dose (30 mg/kg), the experiment was repeated using 30 and 50 mg/kg viloxazine (as 50 mg/kg increased NE, 5-HT, and DA in prior studies). Results: Viloxazine unbound (free drug) plasma concentrations in rats at 30 mg/kg were comparable to free drug concentrations in individuals with ADHD taking clinically effective doses (based on validated population PK models). Viloxazine 30 mg/kg significantly increased extracellular NE, 5-HT, and DA PFC levels compared to vehicle. Concomitant decreases in DHPG, but not 5-HIAA, support the inhibitory effect of viloxazine on NET but not SERT. Conclusion: At clinically relevant concentrations, viloxazine increases PFC NE, DA, and 5-HT. Prefrontal augmentation of 5-HT does not appear to result from 5-HT reuptake inhibition but may be related to activation of 5-HT neurons. The potential therapeutic role of serotonergic effects in ADHD treatment merits further exploration.

Viral vector-mediated upregulation of serine racemase expression in medial prefrontal cortex improves learning and synaptic function in middle age rats.

An age-associated decrease in N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic function contributes to impaired synaptic plasticity and is associated with cognitive impairments. Levels of serine racemase (SR), an enzyme that synthesizes D-serine, an NMDAR co-agonist, decline with age. Thus, enhancing NMDAR function via increased SR expression in middle age, when subtle declines in cognition emerge, was predicted to enhance performance on a prefrontal cortex-mediated task sensitive to aging. Middle-aged (~12 mo) male Fischer-344 rats were injected bilaterally in the medial prefrontal cortex (mPFC) with viral vector (LV), SR (LV-SR) or control (LV-GFP). Rats were trained on the operant attentional set-shift task (AST) to examine cognitive flexibility and attentional function. LV-SR rats exhibited a faster rate of learning compared to controls during visual discrimination of the AST. Extradimensional set shifting and reversal were not impacted. Immunohistochemical analyses demonstrated that LV-SR significantly increased SR expression in the mPFC. Electrophysiological characterization of synaptic transmission in the mPFC slices obtained from LV-GFP and LV-SR animals indicated a significant increase in isolated NMDAR-mediated synaptic responses in LV-SR slices. Thus, results of the current study demonstrated that prefrontal SR upregulation in middle age rats can improve learning of task contingencies for visual discrimination and increase glutamatergic synaptic transmission, including NMDAR activity.

Operationally defining cognitive reserve genes.

Variability in cognitive decline is related to the environment, lifestyle factors, and individual differences in biological aging, including cognitive reserve, plastic properties of the brain, which account for better-than-expected cognition for a given level of brain aging or pathology. Cognitive reserve has not been thoroughly investigated in aged rodents. To address this gap, cognitive reserve was examined using Gene Expression Omnibus data for the CA1 region of the hippocampus of young and aged behaviorally characterized male rats. Statistical filtering identified brain aging and potential cognitive reserve genes, and multiple regression was employed to confirm cognitive reserve genes as genes that predicted better-than-expected cognition for a given level of brain aging. In general, cognitive reserve genes, in which increased expression was associated with better cognition, were not different with age or directly correlated with measures of cognition and appear to act as negative regulators of aging processes, including neuroinflammation and oxidative stress. The results suggest that, for some animals, resilience mechanisms are activated to counteract aging stressors that impair cognition. In contrast, cognitive reserve genes, in which decreased expression was associated with better cognition, were linked to nervous system development and cation transport, suggesting adaptive changes in the circuit to preserve cognition.

Dynamic interplay of frontoparietal cholinergic innervation and cortical reorganization in the regulation of attentional capacities in aging.

Cortical remodeling is linked to age-related cognitive changes in humans; however, the mechanisms underlying cortical reorganization in aging remain unknown. Here we examined the consequences of mild cholinergic thinning of the prefrontal cortex (PFC) and parietal cortex (PC) on attention performance-associated changes in cortical activity in young and aged rats. Prefrontal manipulation produced attentional deficits in aged but not young rats regardless of cholinergic pruning. Stereological assessment of c-fos expression revealed age-related reductions in occipital activity and a corresponding increase in PC activity, but these patterns did not correlate with performance. PC cholinergic deafferentation produced opposite changes in PFC recruitment between young and aged rats. Cholinergic pruning reversed the effects of PFC/PC cholinergic manipulations on the activity of CaMKII- and GAD-positive neurons in aged rats. Our results indicate that cortical shifts depend on multiple factors including chronological age, cholinergic changes, and cortical insult, and that cortical reorganization is not necessarily compensatory. Moreover, the cholinergic system modulates excitation/inhibition homeostasis to improve the efficiency of reorganized cortical circuits and stabilize attentional performance.

Partial microglial depletion is associated with impaired hippocampal synaptic and cognitive function in young and aged rats.

The role of microglia in mediating age-related changes in cognition and hippocampal synaptic function was examined by microglial depletion and replenishment using PLX3397. We observed age-related differences in microglial number and morphology, as well as increased Iba-1 expression, indicating microglial activation. PLX3397 treatment decreased microglial number, with aged rats exhibiting the lowest density. Young rats exhibited increased expression of pro-inflammatory cytokines during depletion and repopulation and maintenance of Iba-1 levels despite reduced microglial number. For aged rats, several cytokines increased with depletion and recovered during repopulation; however, aged rats did not fully recover microglial cell number or Iba-1 expression during repopulation, with a recovery comparable to young control levels rather than aged controls. Hippocampal CA3-CA1 synaptic transmission was impaired with age, and microglial depletion was associated with decreased total synaptic transmission in young and aged rats. A robust decline in N-methyl-d-aspartate-receptor-mediated synaptic transmission arose in young depleted rats specifically. Microglial replenishment normalized depletion-induced synaptic function to control levels; however, recovery of aged animals did not mirror young. Microglial depletion was associated with decreased context-object discrimination memory in both age groups, which recovered with microglial repopulation. Aged rats displayed impaired contextual and cued fear memory, and microglial replenishment did not recover their memory to the level of young. The current study indicates that cognitive function and synaptic transmission benefit from the support of aged microglia and are hindered by removal of these cells. Replenishment of microglia in aging did not ameliorate age-related cognitive impairments or senescent synaptic function.

Longitudinal Characterization and Biomarkers of Age and Sex Differences in the Decline of Spatial Memory.

The current longitudinal study examined factors (sex, physical function, response to novelty, ability to adapt to a shift in light/dark cycle, brain connectivity), which might predict the emergence of impaired memory during aging. Male and female Fisher 344 rats were tested at 6, 12, and 18 months of age. Impaired spatial memory developed in middle-age (12 months), particularly in males, and the propensity for impairment increased with advanced age. A reduced response to novelty was observed over the course of aging, which is inconsistent with cross-sectional studies. This divergence likely resulted from differences in the history of environmental enrichment/impoverishment for cross-sectional and longitudinal studies. Animals that exhibited lower level exploration of the inner region on the open field test exhibited better memory at 12 months. Furthermore, males that exhibited a longer latency to enter a novel environment at 6 months, exhibited better memory at 12 months. For females, memory at 12 months was correlated with the ability to behaviorally adapt to a shift in light/dark cycle. Functional magnetic resonance imaging of the brain, conducted at 12 months, indicated that the decline in memory was associated with altered functional connectivity within different memory systems, most notably between the hippocampus and multiple regions such as the retrosplenial cortex, thalamus, striatum, and amygdala. Overall, some factors, specifically response to novelty at an early age and the capacity to adapt to shifts in light cycle, predicted spatial memory in middle-age, and spatial memory is associated with corresponding changes in brain connectivity. We discuss similarities and differences related to previous longitudinal and cross-sectional studies, as well as the role of sex differences in providing a theoretical framework to guide future longitudinal research on the trajectory of cognitive decline. In addition to demonstrating the power of longitudinal studies, these data highlight the importance of middle-age for identifying potential predictive indicators of sexual dimorphism in the trajectory in brain and cognitive aging.

Effect of Systemic Inflammation on Rat Attentional Function and Neuroinflammation: Possible Protective Role for Food Restriction.

Background: Aging is characterized by subtle cognitive decline, which correlates with increased peripheral inflammation. Acute activation of the peripheral immune system, via lipopolysaccharide (LPS) injection, elicits deficits in hippocampal-dependent spatial memory. Little is known concerning the effect of chronic inflammation on prefrontal cortex (PFC)-dependent vigilance. We examined the impact of repeated LPS injections in young and middle-age rats on the 5-choice serial reaction time task (5-CSRTT), expecting repeated LPS treatment to induce attentional deficits with greater disruption in middle-age. Methods: Male Fischer-344 rats, 4- and 12-months-old, were food restricted and trained on the 5-CSRTT. Once rats reached criterion, they were injected with LPS (1 mg/kg, i.p.) weekly for 4 weeks and testing started 48 h after each injection. To examine the possibility that mild food restriction inherent to the behavioral task influenced inflammation markers, a second group of food-restricted or ad-lib-fed rats was assessed for cytokine changes 48 h after one injection. Results: Performing LPS-treated rats exhibited a sickness response, manifesting as reduced initiated and completed trials during the first week but recovered by the second week of testing. After the first week, LPS-treated rats continued to exhibit longer response latencies, despite no change in food retrieval latency, suggestive of LPS-induced cognitive slowing. Similarly, LPS-induced impairment of attention was observed as increased omissions with heightened cognitive demand and increased age. Repeated LPS-treatment increased the level of PFC IL-1α, and PFC IL-6 was marginally higher in middle-age rats. No effect of age or treatment was observed for plasma cytokines in performing rats. Histological examination of microglia indicated increased colocalization of Iba1+ and CD68+ cells from middle-age relative to young rats. Examination of food restriction demonstrated an attenuation of age- and LPS-related increases in plasma cytokine levels. Conclusions: Systemic inflammation, induced through LPS treatment, impaired attentional function, which was independent of sickness and exacerbated by increased cognitive demand and increased age. Additional studies revealed that food restriction, associated with the task, attenuated markers of neuroinflammation and plasma cytokines. The results emphasize the need for improved methods for modeling low-level chronic systemic inflammation to effectively examine its impact on attention during aging.

Behavior Model for Assessing Decline in Executive Function During Aging and Neurodegenerative Diseases.

Executive dysfunction is a characteristic of several psychiatric and neurodegenerative diseases. Interestingly, executive function, which is mediated by the prefrontal cortex (PFC), commonly declines during aging. The attentional set-shifting task (AST) is commonly and extensively used to assess executive function in rodents, primates, and humans. When properly employed, this task can behaviorally assess attention, response inhibition, and cognitive flexibility. The following section uses research on age-related decline in executive function to demonstrate the methods employed and highlight areas that can confound a study if not employed properly.

Redox Signaling in Neurotransmission and Cognition During Aging.

SIGNIFICANCE: Oxidative stress increases in the brain with aging and neurodegenerative diseases. Previous work emphasized irreversible oxidative damage in relation to cognitive impairment. This research has evolved to consider a continuum of alterations, from redox signaling to oxidative damage, which provides a basis for understanding the onset and progression of cognitive impairment. This review provides an update on research linking redox signaling to altered function of neural circuits involved in information processing and memory. Recent Advances: Starting in middle age, redox signaling triggers changes in nervous system physiology described as senescent physiology. Hippocampal senescent physiology involves decreased cell excitability, altered synaptic plasticity, and decreased synaptic transmission. Recent studies indicate N-methyl-d-aspartate and ryanodine receptors and Ca2+ signaling molecules as molecular substrates of redox-mediated senescent physiology. CRITICAL ISSUES: We review redox homeostasis mechanisms and consider the chemical character of reactive oxygen and nitrogen species and their role in regulating different transmitter systems. In this regard, senescent physiology may represent the co-opting of pathways normally responsible for feedback regulation of synaptic transmission. Furthermore, differences across transmitter systems may underlie differential vulnerability of brain regions and neuronal circuits to aging and disease. FUTURE DIRECTIONS: It will be important to identify the intrinsic mechanisms for the shift in oxidative/reductive processes. Intrinsic mechanism will depend on the transmitter system, oxidative stressors, and expression/activity of antioxidant enzymes. In addition, it will be important to identify how intrinsic processes interact with other aging factors, including changes in inflammatory or hormonal signals. Antioxid. Redox Signal. 28, 1724-1745.

Developmental suppression of forebrain trkA receptors and attentional capacities in aging rats: A longitudinal study.

Basal forebrain (BF) cholinergic neurons innervating the cortex regulate cognitive, specifically attentional, processes. Cholinergic atrophy and cognitive decline occur at an accelerated pace in age-related neurodegenerative disorders such as Alzheimer's disease; however, the mechanism responsible for this phenomenon remains unknown. Here we hypothesized that developmental suppression of nerve growth factor signaling, mediated via tropomyosin-related kinase A (trkA) receptors, would escalate age-related attentional vulnerability. An adeno-associated viral vector expressing trkA shRNA (AAV-trkA) was utilized to knockdown trkA receptors in postnatal rats at an ontogenetic time point when cortical cholinergic inputs mature, and the impact of this manipulation on performance was assessed in animals maintained on an operant attention task throughout adulthood and until old (24 months) age. A within-subject comparison across different time points illustrated a gradual age-related decline in attentional capacities. However, the performance under baseline and distracted conditions did not differ between the AAV-trkA-infused and animals infused with a vector expressing shRNA against the control protein luciferase at any time point. Additional analysis of cholinergic measures conducted at 24 months showed that the capacity of cholinergic terminals to release acetylcholine following a depolarizing stimulus, cortical cholinergic fiber density and BF cholinergic cell size remained comparable between the two groups. Contrary to our predictions, these data indicate that developmental BF trkA disruption does not impact age-related changes in attentional functions. It is possible that life-long engagement in cognitive activity might have potentially rescued the developmental insults on the cholinergic system, thus preserving attentional capacities in advanced age.

Unresponsive Choline Transporter as a Trait Neuromarker and a Causal Mediator of Bottom-Up Attentional Biases.

Some rats [sign-trackers (STs)] are prone to attribute incentive salience to reward cues, which can manifest as a propensity to approach and contact pavlovian cues, and for addiction-like behavior. STs also exhibit poor attentional performance, relative to goal-trackers (GTs), which is associated with attenuated acetylcholine (ACh) levels in prefrontal cortex (Paolone et al., 2013). Here, we demonstrate a cellular mechanism, linked to ACh synthesis, that accounts for attenuated cholinergic capacity in STs. First, we found that electrical stimulation of the basal forebrain increased cortical choline transporter (CHT)-mediated choline transport in GTs, paralleled by a redistribution of CHTs to the synaptic plasma membrane. Neither increases in choline uptake nor translocation of CHTs occurred in STs. Second, and consistent with uptake/translocation alterations, STs demonstrated a reduced ability to support cortical ACh release in vivo compared with GTs after reverse-dialysis to elevate extracellular potassium levels. Third, rats were significantly more likely to develop sign-tracking behavior if treated systemically before pavlovian conditioned approach training with the CHT inhibitor VU6001221. Consistent with its proposed mechanisms, administration of VU6001221 attenuated potassium-evoked ACh levels in prefrontal cortex measured with in vivo microdialysis. We propose that loss of CHT-dependent activation of cortical cholinergic activity in STs degrades top-down executive control over behavior, producing a bias for bottom-up or stimulus-driven attention. Such an attentional bias contributes to nonadaptive reward processing and thus identifies a novel mechanism that can support psychopathology, including addiction.SIGNIFICANCE STATEMENT The vulnerability for addiction-like behavior has been associated with psychological traits, such as the propensity to attribute incentive salience to reward cues that is modeled in rats by sign-tracking behavior. Sign-trackers tend to approach and contact cues associated with reward, whereas their counterparts, the goal-trackers, have a preference for approaching the location of the reward. Here, we show that the capacity of presynaptic cholinergic synapses to respond to stimulation by elevating presynaptic choline uptake and releasing acetylcholine is attenuated in sign-trackers. Furthermore, pharmacological inhibition of choline transport induced sign-tracking behavior. Our findings suggest that reduced levels of cholinergic neuromodulation can mediate an attentional bias toward reward-related cues, thereby allowing such cues to exert relatively greater control over behavior.

Impact of partial dopamine depletion on cognitive flexibility in BDNF heterozygous mice.

RATIONALE: Cognitive flexibility is a key component of executive function and is disrupted in major psychiatric disorders. Brain-derived neurotrophic factor (BDNF) exerts neuromodulatory effects on synaptic transmission and cognitive/affective behaviors. However, the causal mechanisms linking BDNF hypofunction with executive deficits are not well understood. OBJECTIVES: Here, we assessed the consequences of BDNF hemizygosity on cognitive flexibility in mice performing an operant conditioning task. As dopaminergic-glutamatergic interaction in the striatum is important for cognitive processing, and BDNF heterozygous (BDNF(+/-)) mice display a higher dopamine tone in the dorsal striatum, we also assessed the effects of partial striatal dopamine depletion on task performance and glutamate release. RESULTS: BDNF(+/-) mice acquired discrimination learning as well as new rule learning during set-shifting as efficiently as wild-type mice. However, partial removal of striatal dopaminergic inputs with 6-hydroxydopamine (6-OHDA) impaired these cognitive processes by impeding the maintenance of a new learning strategy in both genotypes. BDNF mutants exhibited performance impairments during reversal learning, and these deficits were associated with increased perseveration to the previously acquired strategy. Partial dopamine depletion of the striatum reversed these cognitive impairments. Additionally, reduction in depolarization-evoked glutamate release noted in the dorsal striatum of BDNF(+/-) mice was not observed in 6-OHDA-infused BDNF mutants indicating normalization of glutamatergic transmission in these animals. CONCLUSIONS: Our data illustrate that BDNF signaling regulates cognitive control processes presumably by maintaining striatal dopamine-glutamate balance. Moreover, aberrations in BDNF signaling may act as a common neurobiological substrate that accounts for executive dysfunction observed in multiple psychiatric conditions.

Interactions between Aß oligomers and presynaptic cholinergic signaling: age-dependent effects on attentional capacities.

Substantial evidence suggests that cerebral deposition of the neurotoxic fibrillar form of amyloid precursor protein, ß-amyloid (Aß), plays a critical role in the pathogenesis of Alzheimer's disease (AD). Yet, many aspects of AD pathology including the cognitive symptoms and selective vulnerability of cortically projecting basal forebrain (BF) cholinergic neurons are not well explained by this hypothesis. Specifically, it is not clear why cognitive decline appears early when the loss of BF cholinergic neurons and plaque deposition are manifested late in AD. Soluble oligomeric forms of Aß are proposed to appear early in the pathology and to be better predictors of synaptic loss and cognitive deficits. The present study was designed to examine the impact of Aß oligomers on attentional functions and presynaptic cholinergic transmission in young and aged rats. Chronic intracranial infusions of Aß oligomers produced subtle decrements in the ability of rats to sustain attentional performance with time on task, irrespective of the age of the animals. However, Aß oligomers produced robust detrimental effects on performance under conditions of enhanced attentional load in aged animals. In vivo electrochemical recordings show reduced depolarization-evoked cholinergic signals in Aß-infused aged rats. Moreover, soluble Aß disrupted the capacity of cholinergic synapses to clear exogenous choline from the extracellular space in both young and aged rats, reflecting impairments in the choline transport process that is critical for acetylcholine (ACh) synthesis and release. Although aging per se reduced the cross-sectional area of BF cholinergic neurons and presynaptic cholinergic proteins in the cortex, attentional performance and ACh release remained unaffected in aged rats infused with the control peptide. Taken together, these data suggest that soluble Aß may marginally influence attentional functions at young ages primarily by interfering with the choline uptake processes. However, age-related weakening of the cholinergic system may synergistically interact with these disruptive presynaptic mechanisms to make this neurotransmitter system vulnerable to the toxic effects of oligomeric Aß in robustly impeding attentional capacities.

Cocaine-induced neuroadaptations in the dorsal striatum: glutamate dynamics and behavioral sensitization.

Recent evidence suggests that diminished ability to control cocaine seeking arises from perturbations in glutamate homeostasis in the nucleus accumbens. However, the neurochemical substrates underlying cocaine-induced neuroadaptations in the dorsal striatum and how these mechanisms link to behavioral plasticity is not clear. We employed glutamate-sensitive microelectrodes and amperometry to study the impact of repeated cocaine administration on glutamate dynamics in the dorsolateral striatum of awake freely-moving rats. Depolarization-evoked glutamate release was robustly increased in cocaine-pretreated rats challenged with cocaine. Moreover, the clearance of glutamate signals elicited either by terminal depolarization or blockade of non-neuronal glutamate transporters slowed down dramatically in cocaine-sensitized rats. Repeated cocaine exposure also reduced the neuronal tone of striatal glutamate. Ceftriaxone, a ß-lactam antibiotic that activates the astrocytic glutamate transporter, attenuated the effects of repeated cocaine exposure on synaptic glutamate release and glutamate clearance kinetics. Finally, the antagonism of AMPA glutamate receptors in the dorsolateral striatum blocked the development of behavioral sensitization to repeated cocaine administration. Collectively, these data suggest that repeated cocaine exposure disrupts presynaptic glutamate transmission and transporter-mediated clearance mechanisms in the dorsal striatum. Moreover, such alterations produce an over activation of AMPA receptors in this brain region leading to the sensitized behavioral response to repeated cocaine.

Conditioned inhibition in a rodent model of attention-deficit/hyperactivity disorder.

A deficit in inhibition may underlie some of the symptoms of attention-deficit/hyperactivity disorder (ADHD), particularly impulsivity. However, the data on inhibitory deficits in children with ADHD are mixed. Moreover, there has been little characterization of inhibitory processes in animal models of ADHD. Pavlov's conditioned inhibition procedure allows a direct assessment of the inhibitory status of a stimulus via summation and retardation tests. Therefore, in the current study, we examined conditioned inhibition in spontaneously hypertensive rats (SHRs), the most well-validated animal model of ADHD. SHRs and Wistar rats were trained in a simultaneous feature-negative discrimination in eyeblink conditioning. Each session consisted of a mixture of 2 trial types: a tone paired with a periocular stimulation (A+) or a tone and light presented simultaneously without a periocular stimulation (XA-). Both SHRs and Wistars were able to discriminate A+ from XA- trials. In subsequent summation (X presented simultaneously with a different conditioned excitor, B) and retardation (X paired with the periocular stimulation) tests, the presence of inhibition to X was confirmed in both SHRs and Wistars: X reduced responding to B, and X was slow to develop excitation when paired with periocular stimulation. These results are the first to demonstrate Pavlovian conditioned inhibition in SHRs and to use summation and retardation tests to confirm X as a conditioned inhibitor. The data indicate that conditioned inhibition is intact in SHRs; thus, inhibitory processes that do not require prefrontal cortex or cerebellum may be normal in this strain.