Cortical Acetylcholine Response to Deep Brain Stimulation of the Basal Forebrain.

Background: Deep brain stimulation (DBS), the direct electrical stimulation of neuronal tissue in the basal forebrain to enhance release of the neurotransmitter acetylcholine, is under consideration as a method to improve executive function in patients with dementia. While some small studies indicate a positive response in the clinical setting, the relationship between DBS and acetylcholine pharmacokinetics is incompletely understood. Objective: We examined the cortical acetylcholine response to different stimulation parameters of the basal forebrain. Methods: 2-photon imaging was combined with deep brain stimulation. Stimulating electrodes were implanted in the subpallidal basal forebrain, and the ipsilateral somatosensory cortex was imaged. Acetylcholine activity was determined using the GRABACh-3.0 muscarinic acetylcholine receptor sensor, and blood vessels were imaged with Texas red. Results: Experiments manipulating pulse train frequency demonstrated that integrated acetylcholine induced fluorescence was insensitive to frequency, and that peak levels were achieved with frequencies from 60 to 130 Hz. Altering pulse train length indicated that longer stimulation resulted in higher peaks and more activation with sublinear summation. The acetylcholinesterase inhibitor donepezil increased the peak response to 10s of stimulation at 60Hz, and the integrated response increased 57% with the 2 mg/kg dose, and 126% with the 4 mg/kg dose. Acetylcholine levels returned to baseline with a time constant of 14 to 18 seconds in all experiments. Conclusions: These data demonstrate that acetylcholine receptor activation is insensitive to frequency between 60 and 130 Hz. High peak responses are achieved with up to 900 pulses. Donepezil increases total acetylcholine receptor activation associated with DBS but did not change temporal kinetics. The long time constants observed in the cerebral cortex add to the evidence supporting volume in addition to synaptic transmission.

α7 nicotinic acetylcholine receptors are necessary for basal forebrain activation to increase expression of the nerve growth factor receptor TrkA.

Activation of the basal forebrain leads to increases in the expression of the nerve growth factor receptor, Tropomyosin receptor kinase A (TrkA) and decreases in expression of the beta amyloid cleavage enzyme 1 (BACE1) in the cerebral cortex of both sexes of 5xFAD mice. The studies described in this report were designed to determine if these changes were dependent on acetylcholine receptors. Mice were stimulated unilaterally in the basal forebrain for two weeks. Animals were administered a cholinergic antagonist, or saline, 30 minutes prior to stimulation. Animals administered saline exhibited significant increases in TrkA expression and decreases in BACE1 in the stimulated hemisphere relative to the unstimulated. While both nonselective nicotinic and muscarinic acetylcholine receptor blockade attenuated the BACE1 decline, only the nicotinic receptor antagonism blocked the TrkA increase. Next, we applied selective nicotinic antagonists, and the α7 antagonist blocked the TrkA increases, but the α4ß2 antagonist did not. BACE1 declines were not blocked by either intervention. Mice with a loxP conditional knockout of the gene for the α7 nicotinic receptor were also employed in these studies. Animals were either stimulated bilaterally for two weeks, or left unstimulated. With or without stimulation, the expression of TrkA receptors was lower in the cortical region with the α7 nicotinic receptor knockdown. We thus conclude that α7 nicotinic receptor activation is necessary for normal expression of TrkA and increases caused by basal forebrain activation, while BACE1 declines caused by stimulation have dependency on a broader array of receptor subtypes.

Chronic basal forebrain activation improves spatial memory, boosts neurotrophin receptor expression, and lowers BACE1 and Aß42 levels in the cerebral cortex in mice.

The etiology of Alzheimer's dementia has been hypothesized in terms of basal forebrain cholinergic decline, and in terms of reflecting beta-amyloid neuropathology. To study these different biological elements, we activated the basal forebrain in 5xFAD Alzheimer's model mice and littermates. Mice received 5 months of 1 h per day intermittent stimulation of the basal forebrain, which includes cholinergic projections to the cortical mantle. Then, mice were behaviorally tested followed by tissue analysis. The 5xFAD mice performed worse in water-maze testing than littermates. Stimulated groups learned the water maze better than unstimulated groups. Stimulated groups had 2-3-fold increases in frontal cortex immunoblot measures of the neurotrophin receptors for nerve growth factor and brain-derived neurotrophic factor, and a more than 50% decrease in the expression of amyloid cleavage enzyme BACE1. Stimulation also led to lower Aß42 in 5xFAD mice. These data support a causal relationship between basal forebrain activation and both neurotrophin activation and reduced Aß42 generation and accumulation. The observation that basal forebrain activation suppresses Aß42 accumulation, combined with the known high-affinity antagonism of nicotinic receptors by Aß42, documents bidirectional antagonism between acetylcholine and Aß42.

Development and Characterization of a Micromagnetic Alternative to Cochlear Implant Electrode Arrays.

To stimulate the auditory nerve, cochlear implants directly inject electrical current into surrounding tissue via an implanted electrode array. While many cochlear implant users achieve strong speech perception scores, there remains significant variability. Since cochlear implant electrode arrays are surrounded by a conductive fluid, perilymph, a spread of excitation occurs. The functionality of the cochlea is spatially dependent, and a wider area of excitation negatively affects the hearing of the user. Importantly, magnetic fields are unaffected by the material properties of biological components. To utilize the electromagnetic properties of the human ear, a microcoil array was developed. The microcoils are 4-turn solenoids with a 250- [Formula: see text] turn radius and a 31.75- [Formula: see text] wire radius, coated with Parylene-C. The efficient design was implemented to accelerate testing. The obtained results describe stimulation capabilities. Functionality was validated using a frequency response analyzer to measure how the generated electromagnetic power radiates in space. 99.8% power loss was observed over a 100- [Formula: see text] separation between a pair of identical microcoils. Obtained through finite-element modeling, the microcoils can be driven by a 60 mA, 5 kHz, sinusoidal input for 10 minutes before predicted inflammation. Rattay's activating function was calculated to evaluate the magnetic stimulation effect of external fields on target neurons. Combined with the frequency response analysis, magnitude and spatial effects of the generated potential is established. As a result, each microcoil requires a 400- [Formula: see text]-wide area for each independent stimulation channel, which is 84% narrower than a commercial cochlear array channel, thereby suggesting greater spatial selectivity.

Protocol for behavioral and neural recording during stimulation of the macaque monkey nucleus basalis.

We present an experimental protocol to record neuronal activity during intermittent stimulation of nucleus basalis (NB), as macaque monkeys perform cognitive tasks. This protocol includes implantation of electrodes and generator devices to deliver electrical stimulation to NB using multiple approaches in monkeys. Direct stimulation of NB avoids peripheral cholinergic side effects, optimizes timing, and activates non-cholinergic projection neurons. We describe electrode preparation, surgery, and implantation for direct evaluation of how stimulation affects monkeys' behavior and neuronal activity. For complete details on the use and execution of this profile, please refer to Qi et al. (2021).

Rhythmicity of Prefrontal Local Field Potentials after Nucleus Basalis Stimulation.

The action of acetylcholine in the cortex is critical for cognitive functions and cholinergic drugs can improve functions such as attention and working memory. An alternative means of enhancing cholinergic neuromodulation in primates is the intermittent electrical stimulation of the cortical source of acetylcholine, the nucleus basalis (NB) of Meynert. NB stimulation generally increases firing rate of neurons in the prefrontal cortex, however its effects on single neurons are diverse and complex. We sought to understand how NB stimulation affects global measures of neural activity by recording and analyzing local field potentials (LFPs) in monkeys as they performed working memory tasks. NB stimulation primarily decreased power in the alpha frequency range during the delay interval of working memory tasks. The effect was consistent across variants of the task. No consistent modulation in the delay interval of the task was observed in the gamma frequency range, which has previously been implicated in the maintenance of working memory. Our results reveal global effects of cholinergic neuromodulation via deep brain stimulation, an emerging intervention for the improvement of cognitive function.

Prefrontal cortical plasticity during learning of cognitive tasks.

Training in working memory tasks is associated with lasting changes in prefrontal cortical activity. To assess the neural activity changes induced by training, we recorded single units, multi-unit activity (MUA) and local field potentials (LFP) with chronic electrode arrays implanted in the prefrontal cortex of two monkeys, throughout the period they were trained to perform cognitive tasks. Mastering different task phases was associated with distinct changes in neural activity, which included recruitment of larger numbers of neurons, increases or decreases of their firing rate, changes in the correlation structure between neurons, and redistribution of power across LFP frequency bands. In every training phase, changes induced by the actively learned task were also observed in a control task, which remained the same across the training period. Our results reveal how learning to perform cognitive tasks induces plasticity of prefrontal cortical activity, and how activity changes may generalize between tasks.

Nucleus basalis stimulation enhances working memory by stabilizing stimulus representations in primate prefrontal cortical activity.

Acetylcholine plays a critical role in the neocortex. Cholinergic agonists and acetylcholinesterase inhibitors can enhance cognitive functioning, as does intermittent electrical stimulation of the cortical source of acetylcholine, the nucleus basalis (NB) of Meynert. Here we show in two male monkeys how NB stimulation affects working memory and alters its neural code. NB stimulation increases dorsolateral prefrontal activity during the delay period of spatial working memory tasks and broadens selectivity for stimuli but does not strengthen phasic responses to each neuron's optimal visual stimulus. Paradoxically, despite this decrease in neuronal selectivity, performance improves in many task conditions, likely indicating increased delay period stability. Performance under NB stimulation does decline if distractors similar to the target are presented, consistent with reduced prefrontal selectivity. Our results indicate that stimulation of the cholinergic forebrain increases prefrontal neural activity, and this neuromodulatory tone can improve cognitive performance, subject to a stability-accuracy tradeoff.

Cholinergic Deep Brain Stimulation for Memory and Cognitive Disorders.

Memory and cognitive impairment as sequelae of neurodegeneration in Alzheimer's disease and age-related dementia are major health issues with increasing social and economic burden. Deep brain stimulation (DBS) has emerged as a potential treatment to slow or halt progression of the disease state. The selection of stimulation target is critical, and structures that have been targeted for memory and cognitive enhancement include the Papez circuit, structures projecting to the frontal lobe such as the ventral internal capsule, and the cholinergic forebrain. Recent human clinical and animal model results imply that DBS of the nucleus basalis of Meynert can induce a therapeutic modulation of neuronal activity. Benefits include enhanced activity across the cortical mantle, and potential for amelioration of neuropathological mechanisms associated with Alzheimer's disease. The choice of stimulation parameters is also critical. High-frequency, continuous stimulation is used for movement disorders as a way of inhibiting their output; however, no overexcitation has been hypothesized in Alzheimer's disease and lower stimulation frequency or intermittent patterns of stimulation (periods of stimulation interleaved with periods of no stimulation) are likely to be more effective for stimulation of the cholinergic forebrain. Efficacy and long-term tolerance in human patients remain open questions, though the cumulative experience gained by DBS for movement disorders provides assurance for the safety of the procedure.

Aged rhesus monkeys: Cognitive performance categorizations and preclinical drug testing.

Rodent models have facilitated major discoveries in neurobiology, however, the low success rate of novel medications in clinical trials have led to questions about their translational value in neuropsychiatric drug development research. For age-related disorders of cognition such as Alzheimer' disease (AD) there is interest in moving beyond transgenic amyloid-ß and/or tau-expressing rodent models and focusing more on natural aging and dissociating "healthy" from "pathological" aging to identify new therapeutic targets and treatments. In complex disorders such as AD, it can also be argued that animals with closer neurobiology to humans (e.g., nonhuman primates) should be employed more often particularly in the later phases of drug development. The purpose of the work described here was to evaluate the cognitive capabilities of rhesus monkeys across a wide range of ages in different delayed response tasks, a computerized delayed match to sample (DMTS) task and a manual delayed match to position (DMTP) task. Based on specific performance criteria and comparisons to younger subjects, the older subjects were generally less proficient, however, some performed as well as young subjects, while other aged subjects were markedly impaired. Accordingly, the older subjects could be categorized as aged "cognitively-unimpaired" or aged "cognitively-impaired" with a third group (aged-other) falling in between. Finally, as a proof of principle, we demonstrated using the DMTP task that aged cognitively-impaired monkeys are sensitive to the pro-cognitive effects of a nicotinic acetylcholine receptor (nAChR) partial agonist, encenicline, suggesting that nAChR ligands remain viable as potential treatments for age-related disorders of cognition.

Atomoxetine improves memory and other components of executive function in young-adult rats and aged rhesus monkeys.

Atomoxetine is a norepinephrine reuptake inhibitor and FDA-approved treatment for attention deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults. While there is some evidence that atomoxetine may improve additional domains of cognition beyond attention in both young adults and aged individuals, this subject has not been extensively investigated. Here, we evaluated atomoxetine (in low mg/kg doses) in a variable stimulus duration (vSD) and a variable intertrial interval (vITI) version of the five choice-serial reaction time task (5C-SRTT), and an eight-arm radial arm maze (RAM) procedure in young-adult rats. The compound was further evaluated (in µg/kg-low mg/kg doses) along with nicotine (as a reference compound) and the Alzheimer's disease treatment donepezil in a distractor version of a delayed match to sample task (DMTS-D) in aged monkeys (mean age = 21.8 years). Atomoxetine (depending on the dose) improved accuracy (sustained attention) as well as behaviors related to impulsivity, compulsivity and cognitive inflexibility in both the vSD and vITI tasks and it improved spatial reference memory in the RAM. In the DMTS-D task, both nicotine and atomoxetine, but not donepezil attenuated the effects of the distractor on accuracy at short delays (non-spatial working/short term memory). However, combining sub-effective doses of atomoxetine and donepezil did enhance DMTS-D accuracy indicating the potential of using atomoxetine as an adjunctive treatment with donepezil. Collectively, these animal studies support the further evaluation of atomoxetine as a repurposed drug for younger adults as well older individuals who suffer from deficits in attention, memory and other components of executive function.

Intermittent stimulation in the nucleus basalis of meynert improves sustained attention in rhesus monkeys.

Sustained attention is essential in important behaviors in daily life. Many neuropsychiatric disorders are characterized by a compromised ability to sustain attention, making this cognitive domain an important therapeutic target. In this study, we tested a novel method of improving sustained attention. Monkeys were engaged in a continuous performance task (CPT) while the nucleus basalis of Meynert (NB), the main source of cholinergic innervation of the neocortex, was stimulated. Intermittent NB stimulation improved the animals' performance by increasing the hit rate and decreasing the false alarm rate. Administration of the cholinesterase inhibitor donepezil or the muscarinic antagonist scopolamine alone impaired performance, whereas the nicotinic antagonist mecamylamine alone improved performance. Applying NB stimulation while mecamylamine or donepezil were administered impaired CPT performance. Methylphenidate, a monoaminergic psychostimulant, was applied in conjunction with intermittent stimulation as a negative control, as it does not directly modulate cholinergic output. Methylphenidate also improved performance, and it produced further improvement when combined with NB stimulation. The additive effect of the combination suggested NB stimulation altered behavior independently from methylphenidate effects. We conclude that basal forebrain projections contribute to sustained attention, and that intermittent NB stimulation is an effective way of improving performance.

Potential for intermittent stimulation of nucleus basalis of Meynert to impact treatment of alzheimer's disease.

The brain's cholinergic arousal pathways decline in parallel with the brain's executive functions in aging and Alzheimer's Disease. The frontline and currently most effective approach to treating Alzheimer's disease is the administration of cholinesterase inhibitors, which, in a dose dependent manner, improve the symptoms of cognitive decline over the first months of treatment before further decline occurs. We recently showed that intermittent deep brain stimulation of the nucleus basalis of Meynert improves working memory function in young adult monkeys, and that this improvement depended on cholinergic function. Within minutes, the monkeys' ability to remember stimuli over a delay period improved. Over months, the monkeys performed the working memory task better even in the absence of stimulation. Here, we show historical data from our monkey colony in which more than two dozen animals have performed the same behavioral task to asymptotic performance levels. Using a distribution based on our historical data, we estimate that the monkeys receiving intermittent stimulation leapt over the performance level of 32-44 percent of peer animals in the first several months after stimulation was initiated. Implications for a parallel increase in cognitive function for early Alzheimer's patients are discussed.

Intermittent Stimulation of the Nucleus Basalis of Meynert Improves Working Memory in Adult Monkeys.

Acetylcholine in the neocortex is critical for executive function [1-3]. Degeneration of cholinergic neurons in aging and Alzheimer's dementia is commonly treated with cholinesterase inhibitors [4-7]; however, these are modestly effective and are associated with side effects that preclude effective dosing in many patients [8]. Electrical activation of the nucleus basalis (NB) of Meynert, the source of neocortical acetylcholine [9, 10], provides a potential method of improving cholinergic activation [11, 12]. Here we tested whether NB stimulation would improve performance of a working memory task in a nonhuman primate model. Unexpectedly, intermittent stimulation proved to be most beneficial (60 pulses per second, for 20 s every minute), whereas continuous stimulation often impaired performance. Pharmacological experiments confirmed that the effects depended on cholinergic activation. Donepezil, a cholinesterase inhibitor, restored performance in animals impaired by continuous stimulation but did not improve performance further during intermittent stimulation. Intermittent stimulation was rendered ineffective by either nicotinic or muscarinic receptor antagonists. In the months after stimulation began, performance also improved in sessions without stimulation. Our results reveal that intermittent NB stimulation can improve working memory, a finding that has implications for restoring cognitive function in aging and Alzheimer's dementia.

Network Supervision of Adult Experience and Learning Dependent Sensory Cortical Plasticity.

The brain is capable of remodeling throughout life. The sensory cortices provide a useful preparation for studying neuroplasticity both during development and thereafter. In adulthood, sensory cortices change in the cortical area activated by behaviorally relevant stimuli, by the strength of response within that activated area, and by the temporal profiles of those responses. Evidence supports forms of unsupervised, reinforcement, and fully supervised network learning rules. Studies on experience-dependent plasticity have mostly not controlled for learning, and they find support for unsupervised learning mechanisms. Changes occur with greatest ease in neurons containing α-CamKII, which are pyramidal neurons in layers II/III and layers V/VI. These changes use synaptic mechanisms including long term depression. Synaptic strengthening at NMDA-containing synapses does occur, but its weak association with activity suggests other factors also initiate changes. Studies that control learning find support of reinforcement learning rules and limited evidence of other forms of supervised learning. Behaviorally associating a stimulus with reinforcement leads to a strengthening of cortical response strength and enlarging of response area with poor selectivity. Associating a stimulus with omission of reinforcement leads to a selective weakening of responses. In some preparations in which these associations are not as clearly made, neurons with the most informative discharges are relatively stronger after training. Studies analyzing the temporal profile of responses associated with omission of reward, or of plasticity in studies with different discriminanda but statistically matched stimuli, support the existence of limited supervised network learning. © 2017 American Physiological Society. Compr Physiol 7:977-1008, 2017.

Tone identification behavior in Rattus norvegicus: muscarinic receptor blockage lowers responsiveness in nontarget selective neurons, while nicotinic receptor blockage selectively lowers target responses.

Learning to associate a stimulus with reinforcement causes plasticity in primary sensory cortex. Neural activity caused by the associated stimulus is paired with reinforcement, but population analyses have not found a selective increase in response to that stimulus. Responses to other stimuli increase as much as, or more than, responses to the associated stimulus. Here, we applied population analysis at a new time point and additionally evaluated whether cholinergic receptor blockers interacted with the plastic changes in cortex. Three days of tone identification behavior caused responsiveness to increase broadly across primary auditory cortex, and target responses strengthened less than overall responsiveness. In pharmacology studies, behaviorally impairing doses of selective acetylcholine receptor blockers were administered during behavior. Neural responses were evaluated on the following day, while the blockers were absent. The muscarinic group, blocked by scopolamine, showed lower responsiveness and an increased response to the tone identification target that exceeded both the 3-day control group and task-naïve controls. Also, a selective increase in the late phase of the response to the tone identification stimulus emerged. Nicotinic receptor antagonism, with mecamylamine, more modestly lowered responses the following day and lowered target responses more than overall responses. Control acute studies demonstrated the muscarinic block did not acutely alter response rates, but the nicotinic block did. These results lead to the hypothesis that the decrease in the proportion of the population spiking response that is selective for the target may be explained by the balance between effects modulated by muscarinic and nicotinic receptors.

The most sensitive inputs to cutaneous representing regions of primary somatosensory cortex do not change with behavioral training.

Learning a sensory detection task leads to an increased primary sensory cortex response to the detected stimulus, while learning a sensory discrimination task additionally leads to a decreased sensory cortex response to the distractor stimulus. Neural responses are scaled up, and down, in strength, along with concomitant changes in receptive field size. The present work considers neural response properties that are invariant to learning. Data are drawn from two animals that were trained to detect and discriminate spatially separate taps delivered to positions on the skin of their fingers. Each animal was implanted with electrodes positioned in area 3b, and responses were derived on a near daily basis over 84 days in animal 1 and 202 days in animal 2. Responses to taps delivered in the receptive field were quantitatively measured each day, and receptive fields were audiomanually mapped each day. In the subset of responses that had light cutaneous receptive fields, a preponderance of the days, the most sensitive region of the field was invariant to training. This skin region was present in the receptive field on all, or nearly all, occasions in which the receptive field was mapped, and this region constituted roughly half of the most sensitive region. These results suggest that maintaining the most sensitive inputs as dominant in cortical receptive fields provide a measure of stability that may be transformationally useful for minimizing reconstruction errors and perceptual constancy.