Cholinergic Input Research Articles

Postnatal development of cholinergic input to the thalamic reticular nucleus of the mouse.

The thalamic reticular nucleus (TRN), a shell-like structure comprised of GABAergic neurons, gates signal transmission between thalamus and cortex. While TRN is innervated by axon collaterals of thalamocortical and corticothalamic neurons, other ascending projections modulate activity during different behavioral states such as attention, arousal, and sleep-wake cycles. One of the largest arise from cholinergic neurons of the basal forebrain and brainstem. Despite its integral role, little is known about how or when cholinergic innervation and synapse formation occurs. We utilized genetically modified mice, which selectively express fluorescent protein and/or channelrhodopsin-2 in cholinergic neurons, to visualize and stimulate cholinergic afferents in the developing TRN. Cholinergic innervation of TRN follows a ventral-to-dorsal progression, with nonvisual sensory sectors receiving input during week 1, and the visual sector during week 2. By week 3, the density of cholinergic fibers increases throughout TRN and forms a reticular profile. Functional patterns of connectivity between cholinergic fibers and TRN neurons progress in a similar manner, with weak excitatory nicotinic responses appearing in nonvisual sectors near the end of week 1. By week 2, excitatory responses become more prevalent and arise in the visual sector. Between weeks 3-4, inhibitory muscarinic responses emerge, and responses become biphasic, exhibiting a fast excitatory, and a long-lasting inhibitory component. Overall, the development of cholinergic projections in TRN follows a similar plan as the rest of sensory thalamus, with innervation of nonvisual structures preceding visual ones, and well after the establishment of circuits conveying sensory information from the periphery to the cortex.

Chloride Influx of Anion Exchanger 2 Was Modulated by Calcium-Dependent Spinophilin in Submandibular Glands.

Secretory glands including salivary glands by many hormonal inputs produce and secrete biological fluids determined by variety of ion transporters. Spinophilin is a multifunctional scaffolding protein, which involved in receptor signaling and regulation of anion exchangers AE2 activity. We found that spinophilin expressed in salivary glands. The role of salivary spinophilin on the modulation of chloride/bicarbonate exchange remains unknown. The spinophilin enhanced AE2 activity and associated with a STE20/SPS1-related kinase and showed an additive effect on the modulation of the activity of AE2. The cholinergic stimulation and subsequent intracellular Ca2+ increase was required for the interaction with AE2 and spinophilin and abrogated the enhanced effect of spinophilin on Cl− transporting activity. Ductal chloride/bicarbonate exchange activity was increased in pretreatment with carbachol. The CaMKII inhibitor KN-93 suppressed the chloride/bicarbonate exchange activity of ducts, suggesting that CaMKII was required for ductal chloride/bicarbonate exchange activity. Additionally, microtubule destabilization by nocodazole attenuated the interaction of AE2 and spinophilin and almost abolished the ductal chloride/bicarbonate exchange activity. The treatment of siRNA-spinophilin on the isolated salivary ducts also reduced the ductal chloride/bicarbonate exchange activity. Therefore, role of salivary spinophilin on AE2 may facilitate the Cl− influx from basolateral in salivary glands in response to cholinergic inputs.

Vocal learning in songbirds requires cholinergic signaling in a motor cortex-like nucleus.

Cholinergic inputs to cortex modulate plasticity and sensory processing, yet little is known about their role in motor control. Here, we show that cholinergic signaling in a songbird vocal motor cortical area, the robust nucleus of the arcopallium (RA), is required for song learning. Reverse microdialysis of nicotinic and muscarinic receptor antagonists into RA in juvenile birds did not significantly affect syllable timing or acoustic structure during vocal babbling. However, chronic blockade over weeks reduced singing quantity and impaired learning, resulting in an impoverished song with excess variability, abnormal acoustic features, and reduced similarity to tutor song. The demonstration that cholinergic signaling in a motor cortical area is required for song learning motivates the songbird as a tractable model system to identify roles of the basal forebrain cholinergic system in motor control. NEW & NOTEWORTHY Cholinergic inputs to cortex are evolutionarily conserved and implicated in sensory processing and synaptic plasticity. However, functions of cholinergic signals in motor areas are understudied and poorly understood. Here, we show that cholinergic signaling in a songbird vocal motor cortical area is not required for normal vocal variability during babbling but is essential for developmental song learning. Cholinergic modulation of motor cortex is thus required for learning but not for the ability to sing.

1. The development of propagated discharge and behavioural arrest in hippocampal and amygdala kindled seizures

Patients with focal temporal lobe seizures often experience transient episodes of impaired awareness with behavioural arrest, but the precise mechanism remains unknown. The Blumenfeld hypothesis attributes these deficits to a loss of cholinergic input to the cortex. This is presumed to result from increased activation of inhibitory regions that suppress subcortical arousal, giving rise to slow wave activity. To investigate this hypothesis – and more specifically, to characterize the relationship between propagated discharge, cortical slow waves and behavioural arrest – we performed kindling studies in rats. We found that seizure discharge took longer to spread from the amygdala than the hippocampus, and took more kindling stimulations to elicit behavioural arrest. In addition, the onset of propagated discharge in subcortical and cortical sites did not always match with the onset of behavioural arrest. Importantly, the activity seen in the cortex did not resemble the slow waves seen in deep sleep. Together, these findings suggest an additional mechanism – other than the Blumenfeld hypothesis – to explain how temporal lobe seizures may produce behavioural arrest and impair awareness.

Longitudinal Alzheimer’s Degeneration Reflects the Spatial Topography of Cholinergic Basal Forebrain Projections

The cholinergic neurons of the basal forebrain (BF) provide virtually all of the brain's cortical and amygdalar cholinergic input. They are particularly vulnerable to neuropathology in early Alzheimer's disease (AD) and may trigger the emergence of neuropathology in their cortico-amygdalar projection system through cholinergic denervation and trans-synaptic spreading of misfolded proteins. We examined whether longitudinal degeneration within the BF can explain longitudinal cortico-amygdalar degeneration in older human adults with abnormal cerebrospinal fluid biomarkers of AD neuropathology. We focused on two BF subregions, which are known to innervate cortico-amygdalar regions via two distinct macroscopic cholinergic projections. To further assess whether structural degeneration of these regions in AD reflects cholinergic denervation, we used the [18F] FEOBV radiotracer, which binds to cortico-amygdalar cholinergic terminals. We found that the two BF subregions explain spatially distinct patterns of cortico-amygdalar degeneration, which closely reflect their cholinergic projections, and overlap with [18F] FEOBV indices of cholinergic denervation.

Anti-inflammatory Cholinergic Signals Inhibit Islet Resident Macrophage Responses to ATP in Living Pancreatic Tissue Slices

When stimulated by high glucose concentrations, beta cells release ATP together with insulin. Under these conditions, the ATP concentration in the islet reaches levels that would induce inflammatory responses in other tissues. Yet inflammation does not occur during normal islet physiology. We therefore postulated that there is a local anti-inflammatory circuit that prevents pathological immune responses. Our studies focused on resident macrophages, which are the local immune cells in the pancreatic islet. We recently showed that islet macrophages detect ATP to gauge the level of beta cell activation. We hypothesized that the potentially inflammatory responses to ATP are inhibited in macrophages by cholinergic input from parasympathetic nerves, as reported for other organs. Using immunohistochemistry, we found that most islet resident macrophages were indeed contacted by local cholinergic nerves, with an innervation density that was equivalent to that of beta cells. To study the impact of local cholinergic nerves on resident macrophages, we used living tissue pancreatic slices, where activity of islet cells, nerves, and macrophages can be visualized in a preserved local microenvironment. We determined that Ca2+ responses to ATP were inhibited in islet macrophages by nicotine, an agonist of nicotinic acetylcholine receptors. These results indicate that macrophages receive cholinergic input from parasympathetic nerves. We expect our studies to identify parasympathetic nerves as a source of anti-inflammatory signals that efficiently dampen the potentially pro-inflammatory environment in the islet. Disclosure J. Weitz: None. R. Rodriguez Diaz: None. J. Almaca: None. M. Makhmutova: None. A. Caicedo: None.

Cholinergic excitation complements glutamate in coding visual information in retinal ganglion cells.

Starburst amacrine cells release GABA and ACh. This study explores the coordinated function of starburst-mediated cholinergic excitation and GABAergic inhibition to bistratified retinal ganglion cells, predominantly direction-selective ganglion cells (DSGCs). In rat retina, under our recording conditions, starbursts were found to provide the major excitatory drive to a sub-population of ganglion cells whose dendrites co-stratify with starburst dendrites (putative DSGCs). In mouse retina, recordings from genetically identified DSGCs at physiological temperatures reveal that ACh inputs dominate the response to small spot-high contrast light stimuli, with preferential addition of bipolar cell input shifting the balance towards glutamate for larger spot stimuli In addition, starbursts also appear to gate glutamatergic excitation to DSGCs by postsynaptic and possibly presynaptic inhibitory processes ABSTRACT: Starburst amacrine cells release both GABA and ACh, allowing them to simultaneously mediate inhibition and excitation. However, the precise pre- and postsynaptic targets for ACh and GABA remain under intense investigation. Most previous studies have focused on starburst-mediated postsynaptic GABAergic inhibition and its role in the formation of directional selectivity in ganglion cells. However, the significance of postsynaptic cholinergic excitation is only beginning to be appreciated. Here, we found that light-evoked responses measured in bi-stratified rat ganglion cells with dendrites that co-fasciculate with ON and OFF starburst dendrites (putative direction-selective ganglion cells, DSGCs) were abolished by the application of nicotinic receptor antagonists, suggesting ACh could act as the primary source of excitation. Recording from genetically labelled DSGCs in mouse retina at physiological temperatures revealed that cholinergic synaptic inputs dominated the excitation for high contrast stimuli only when the size of the stimulus was small. Canonical glutamatergic inputs mediated by bipolar cells were prominent when GABA/glycine receptors were blocked or when larger spot stimuli were utilized. In mouse DSGCs, bipolar cell excitation could also be unmasked through the activation of mGluR2,3 receptors, which we show suppresses starburst output, suggesting that GABA from starbursts serves to inhibit bipolar cell signals in DSGCs. Taken together, these results suggest that starbursts amplify excitatory signals traversing the retina, endowing DSGCs with the ability to encode fine spatial information without compromising their ability to encode direction.

Effects of Cholinergic Lesions and Cholinesterase Inhibitors on Aromatase and Estrogen Receptor Expression in Different Regions of the Rat Brain

Cholinergic projections have been shown to interact with estrogens in ways that influence synaptic plasticity and cognitive performance. The mechanisms are not well understood. The goal of this study was to investigate whether cholinergic projections influence brain estrogen production by affecting aromatase (ARO), or influence estrogen signaling by affecting estrogen receptor expression. In the first experiment, ovariectomized rats received intraseptal injection of the selective immunotoxin 192IgG-saporin to destroy cholinergic inputs to the hippocampus. In the second experiment ovariectomized rats received daily intraperitoneal injections of the cholinesterase inhibitors donepezil or galantamine for 1 week. ARO activity and relative levels of ARO, ERα, ERß, and GPR30 mRNAs were quantified in the hippocampus, frontal cortex, amygdala and preoptic area. Results show that the cholinergic lesions effectively removed cholinergic inputs to the hippocampus, but had no significant effect on ARO or on relative levels of ER mRNAs. Likewise, injections of the cholinesterase inhibitors had no effect on ARO or ER expression in most regions of the brain. This suggests that effects of cholinergic inputs on synaptic plasticity and neuronal function are not mediated by effects on local estrogen production or ER expression. One exception was the amygdala where treating with galantamine was associated with a significant increase in ARO activity. The amygdala is a key structure involved in registering fear and anxiety. Hence this finding may be clinically relevant to elderly patients who are treated for memory impairment and who also struggle with fear and anxiety disorders.

Disruption of medial septum and diagonal bands of Broca cholinergic projections to the ventral hippocampus disrupt auditory fear memory.

In classical fear conditioning, a neutral conditioned stimulus (CS) is paired with an aversive unconditioned stimulus (US), which leads to a fear memory. If the CS is repeatedly presented without the US after fear conditioning, the formation of an extinction memory occurs, which inhibits fear memory expression. A previous study has demonstrated that selective cholinergic lesions in the medial septum and vertical limb of the diagonal bands of Broca (MS/vDBB) prior to fear and extinction learning disrupt contextual fear memory discrimination and acquisition of extinction memory. MS/vDBB cholinergic neurons project to a number of substrates that are critical for fear and extinction memory. However, it is currently unknown which of these efferent projections are critical for contextual fear memory discrimination and extinction memory. To address this, we induced cholinergic lesions in efferent targets of MS/vDBB cholinergic neurons. These included the dorsal hippocampus (dHipp), ventral hippocampus (vHipp), medial prefrontal cortex (mPFC), and in the mPFC and dHipp combined. None of these lesion groups exhibited deficits in contextual fear memory discrimination or extinction memory. However, vHipp cholinergic lesions disrupted auditory fear memory. Because MS/vDBB cholinergic neurons are the sole source of acetylcholine in the vHipp, these results suggest that MS/vDBB cholinergic input to the vHipp is critical for auditory fear memory. Taken together with previous findings, the results of this study suggest that MS/vDBB cholinergic neurons are critical for fear and extinction memory, though further research is needed to elucidate the role of MS/vDBB cholinergic neurons in these types of emotional memory.

Synaptic Release of Acetylcholine Rapidly Suppresses Cortical Activity by Recruiting Muscarinic Receptors in Layer 4.

Cholinergic afferents from the basal forebrain (BF) can influence cortical activity on rapid time scales, enabling sensory information processing and exploratory behavior. However, our understanding of how synaptically released acetylcholine (ACh) influences cellular targets in distinct cortical layers remains incomplete. Previous studies have shown that rapid changes in cortical dynamics induced by phasic BF activity can be mediated by the activation of nicotinic ACh receptors (nAChRs) expressed in distinct types of GABAergic interneurons. In contrast, muscarinic ACh receptors (mAChRs) are assumed to be involved in slower and more diffuse ACh signaling following sustained increases in afferent activity. Here, we examined the mechanisms underlying fast cholinergic control of cortical circuit dynamics by pairing optical stimulation of cholinergic afferents with evoked activity in somatosensory cortical slices of mice of either sex. ACh release evoked by single stimuli led to a rapid and persistent suppression of cortical activity, mediated by mAChRs expressed in layer 4 and to a lesser extent, by nAChRs in layers 1-3. In agreement, we found that cholinergic inputs to layer 4 evoked short-latency and long-lasting mAChR-dependent inhibition of the large majority of excitatory neurons, whereas inputs to layers 1-3 primarily evoked nAChR-dependent excitation of different classes of interneurons. Our results indicate that the rapid cholinergic control of cortical network dynamics is mediated by both nAChRs and mAChRs-dependent mechanisms, which are expressed in distinct cortical layers and cell types.SIGNIFICANCE STATEMENT Acetylcholine (ACh) release from basal forebrain (BF) afferents to cortex influences a variety of cognitive functions including attention, sensory processing, and learning. Cholinergic control occurs on the time scale of seconds and is mediated by BF neurons that generate action potentials at low rates, indicating that ACh acts as a point-to-point neurotransmitter. Our findings highlight that even brief activation of cholinergic afferents can recruit both nicotinic and muscarinic ACh receptors expressed in several cell types, leading to modulation of cortical activity on distinct time scales. Furthermore, they indicate that the initial stages of cortical sensory processing are under direct cholinergic control.

Effects of systemic cholinergic antagonism on reinforcer devaluation in macaques

The capacity to adjust actions based on new information is a vital cognitive function. An animal’s ability to adapt behavioral responses according to changes in reward value can be measured using a reinforcer devaluation task, wherein the desirability of a given object is reduced by decreasing the value of the associated food reinforcement. Elements of the neural circuits serving this ability have been studied in both rodents and nonhuman primates. Specifically, the basolateral amygdala, orbitofrontal cortex, nucleus accumbens, and mediodorsal thalamus have each been shown to play a critical role in the process of value updating, required for adaptive goal selection. As these regions receive dense cholinergic input, we investigated whether systemic injections of non-selective nicotinic or muscarinic acetylcholine receptor antagonists, mecamylamine and scopolamine, respectively, would impair performance on a reinforcer devaluation task. Here we demonstrate that in the presence of either a nicotinic or muscarinic antagonist, animals are able to shift their behavioral responses in an appropriate manner, suggesting that disruption of cholinergic neuromodulation is not sufficient to disrupt value updating, and subsequent goal selection, in rhesus macaques.

Pharmacological Modulation of Long-Term Potentiation-Like Activity in the Dorsolateral Prefrontal Cortex.

Background: Long-term potentiation (LTP) depends on glutamatergic neurotransmission and is modulated by cholinergic, dopaminergic and GABAergic inputs. Paired associative stimulation (PAS) is a neurostimulation paradigm that, when combined with electroencephalography (EEG), assesses LTP-like activity (PAS-induced LTP) in the dorsolateral prefrontal cortex (DLPFC). Thus, we conducted a study to assess the role of cholinergic, dopaminergic, GABAergic and glutamatergic neurotransmission on PAS-induced LTP in the DLPFC. We hypothesized that increasing the dopaminergic tone with L-DOPA and the cholinergic tone with rivastigmine will enhance PAS-induced LTP, while increasing the GABAergic tone with baclofen and inhibiting glutamatergic neurotransmission with dextromethorphan will reduce it compared to placebo.Methods: In this randomized controlled, double-blind cross-over within-subject study, 12 healthy participants received five sessions of PAS to the DLPFC in a random order, each preceded by the administration of placebo or one of the four active drugs. PAS-induced LTP was assessed after each drug administration and compared to PAS-induced LTP after placebo.Results: As predicted, L-DOPA and rivastigmine resulted in enhanced PAS-induced LTP in the DLPFC and dextromethorphan inhibited it compared to placebo. In contrast, baclofen did not significantly suppress PAS-induced LTP compared to placebo.Conclusions: This study provides a novel approach to study DLPFC neuroplasticity and its modulation in patients with brain disorders that are associated with abnormalities in these neurochemical systems. This study was based on a single dose administration of each drug. Given that these drugs are typically administered chronically, future studies should assess the effects of chronic administration.

Role of ion channels in the functional response of conjunctival goblet cells to dry eye.

Optimal vision requires an ocular surface with a stable tear film whose many critical tasks include providing >70% of the eye's refractive power. However, for millions, tear film instability produces uncomfortable sight-impairing dry eye. Despite the multitude of etiologies for dry eye, a universal hallmark is hyperosmolarity of the tear film. Presently, knowledge of how the ocular surface responds to hyperosmolarity remains incomplete with little understood about the role of ion channels. This bioelectric analysis focused on conjunctival goblet cells whose release of tear-stabilizing mucin is a key adaptive response to dry eye. In freshly excised rat conjunctiva, perforated-patch recordings demonstrated that a ≥10% rise in osmolarity triggers goblet cells to rapidly generate a ~15-mV hyperpolarization due to the oxidant-dependent activation of ATP-sensitive K+ (KATP) channels. High-resolution membrane capacitance measurements used to monitor exocytosis revealed that this hyperpolarization results in an approximately fourfold boost in exocytotic activity evoked by cholinergic input, which in vivo occurs via a neural reflex and depends chiefly on calcium influxing down its electro-gradient. We discovered that this adaptive response is transient. During 30-80 min of hyperosmolarity, development of a depolarizing nonspecific cation conductance fully counterbalances the KATP-driven hyperpolarization and thereby eliminates the exocytotic boost. We conclude that hyperosmotic-induced hyperpolarization is a previously unappreciated mechanism by which goblet cells respond to transient ocular dryness. Loss of this voltage increase during long-term dryness/hyperosmolarity may account for the clinical conundrum that goblet cells in chronically dry eyes can remain filled with mucin even though the tear film is hyperosmotic and mucin-deficient.

Cholinergic Hypofunction in Presbycusis-Related Tinnitus With Cognitive Function Impairment: Emerging Hypotheses.

Presbycusis (age-related hearing loss) is a potential risk factor for tinnitus and cognitive deterioration, which result in poor life quality. Presbycusis-related tinnitus with cognitive impairment is a common phenotype in the elderly population. In these individuals, the central auditory system shows similar pathophysiological alterations as those observed in Alzheimer’s disease (AD), including cholinergic hypofunction, epileptiform-like network synchronization, chronic inflammation, and reduced GABAergic inhibition and neural plasticity. Observations from experimental rodent models indicate that recovery of cholinergic function can improve memory and other cognitive functions via acetylcholine-mediated GABAergic inhibition enhancement, nicotinic acetylcholine receptor (nAChR)-mediated anti-inflammation, glial activation inhibition and neurovascular protection. The loss of cholinergic innervation of various brain structures may provide a common link between tinnitus seen in presbycusis-related tinnitus and age-related cognitive impairment. We hypothesize a key component of the condition is the withdrawal of cholinergic input to a subtype of GABAergic inhibitory interneuron, neuropeptide Y (NPY) neurogliaform cells. Cholinergic denervation might not only cause the degeneration of NPY neurogliaform cells, but may also result in decreased AChR activation in GABAergic inhibitory interneurons. This, in turn, would lead to reduced GABA release and inhibitory regulation of neural networks. Reduced nAChR-mediated anti-inflammation due to the loss of nicotinic innervation might lead to the transformation of glial cells and release of inflammatory mediators, lowering the buffering of extracellular potassium and glutamate metabolism. Further research will provide evidence for the recovery of cholinergic function with the use of cholinergic input enhancement alone or in combination with other rehabilitative interventions to reestablish inhibitory regulation mechanisms of involved neural networks for presbycusis-related tinnitus with cognitive impairment.

Evaluating the Effects of Cholinergic Depletion in the PPTg, LDTg, and VTA on Acoustic Startle Response and Morphine‐Related Reward

Opiate abuse is a worldwide epidemic that costs nations billions of dollars to treat. Rewarding effects of opioids and other drugs of abuse are partially reliant on the activity of dopaminergic neurons located in reward centers of the brain such as the ventral tegmental area (VTA). The VTA receives cholinergic input from only two sources, the pedunculopontine tegmentum (PPTg) and the laterodorsal tegmentum (LDTg). Excitotoxic lesions to the PPTg in rats have previously been shown to reduce the rewarding effect of opiates (Bechara & van der Kooy, 1989; Olmstead & Franklin, 1997; Parker & van der Kooy, 1995). These studies were unable to identify the specific neuronal subtype responsible for the reduced rewarding effect, though they suggested the cholinergic neurons were responsible. By using a diphtheria‐UII fusion toxin to selectively ablate cholinergic neurons in the PPTg and LDTg we were able to test the role of cholinergic projections to the VTA. The toxin was injected bilaterally into the PPTg, LDTg, or VTA of male Sprague‐Dawley rats through stereotaxic surgery. Injection into the VTA creates a retrograde lesion that specifically ablates the cholinergic neurons of the PPTg and LDTg that innervate the VTA.The animals were subjected to an acoustic startle reflex/pre‐pulse inhibition paradigm in weeks 4, 8, and 10 post‐surgery. Animals with cholinergic depletion in the PPTg were unable to startle to the point where pre‐pulse inhibition was unable to be measured. While not significant, there was a trend toward an increase in startle in animals that received a VTA retrograde lesion. These findings suggest that the cholinergic PPTg is necessary for integrating sensory processing and motor output. Cholinergic input to the VTA from both the PPTg and LDTg produced a novel effect. Ten weeks post‐surgery the rats were subjected to a morphine conditioned place preference paradigm (CPP), an extinction period, and then a cocaine CPP paradigm. Animals with lesions to the LDTg were able to form cocaine‐mediated CPP but not morphine‐mediated CPP. PPTg lesioned animals formed preference for both morphine and cocaine. Sham animals and the VTA retrograde animals also formed preference for both drugs. These findings suggest that cholinergic neurons in the LDTg that project to an area that is not the VTA likely play a role in morphine‐related reward, and that the cholinergic output from the PPTg is not required in the formation of morphine conditioned place preference. Taking the results of both experiments together, it can be inferred that the cholinergic LDTg plays a role in reward while the cholinergic PPTg plays a role in sensory processing. It is likely that these two areas work in concert to modulate a variety of behaviors. While these findings don't provide a treatment for opiate abuse, they help to further our understanding of the neuronal mechanisms that drive drug addiction.Support or Funding InformationR00DA024754This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Plasticity of sympathetic post‐ganglionic neuron after spinal cord injury

Autonomic dysreflexia (AD) is a hypertensive crisis seen in high‐thoracic spinal cord injured (ht‐SCI) patients normally triggered by noxious stimuli below the injury level. Previous animal research on mechanisms underlying AD have mostly focused on neural plasticity within the injured spinal cord. No studies have assessed whether there is also plasticity in the sympathetic post‐ganglionic neurons (SPNs) ‐ the final neural element responsible for vasoconstriction.Thoracic sympathetic post‐ganglionic neurons (tSPNs) are located in paravertebral ganglia and provide the dominant sympathetic control of blood pressure. Their cellular properties are understudied. The few previous ex vivo studies used sharp electrodes that likely leads to significant impalement injury‐induced changes [1, 2]. We undertook whole‐cell patch clamp recordings in an adult mouse tSPN ex vivo preparation. Compared to earlier reports using sharp electrodes, we observed an order of magnitude greater passive membrane properties (cell resistance and membrane time constant) and repetitive rather than phasic firing properties. Together these observations support much greater excitability and integrative actions of tSPNs than previously assumed.Using the same methodology we then studied whether these neurons undergo plastic changes after high‐thoracic (T2) spinal cord transection (ht‐SCI), an animal model of AD. Recordings were obtained from T3‐T9 tSPNs at 3 weeks and 6 weeks post injury. Preliminary data suggest that early after injury (3 weeks), tSPNs are hypo‐active compared to normal animal, including more hyperpolarized resting membrane potential and higher rheobase and threshold voltage. At 6 weeks post‐SCI, tSPN passive membrane properties are similar to control mice. However, several neurons show spontaneously unstable membrane potential (55% of recorded neurons), which is rarely seen in control. Evidences that tSPNs may become more excitable include lower rheobase and threshold voltage values on average and an increased incidence of nifedipine sensitive persistent inward currents (PIC; 69% of recorded neurons after SCI has PIC comparing to 22% in control). Also, tSPNs appear to receive more frequent spontaneous cholinergic synaptic input after SCI.Support or Funding InformationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MICROINJECTION OF UROTENSIN II INTO THE PEDUNCULOPONTINE TEGMENTNUM LEADS TO AN INCREASED PERCEPTION OF SENSORY STIMULI

The pedunculopontine tegmentum (PPTg) plays a role in processing multiple sensory inputs and innervates brain regions associated with reward‐related behaviors. The urotensin II receptor, activated by the urotensin II peptide (UII), is selectively expressed by the cholinergic neurons of the PPTg. Although the exact function of cholinergic neurons of the PPTg are unknown, they are thought to modify the perception of reward magnitude or salience detection. We hypothesized that the activation of PPTg cholinergic neurons would alter sensory processing across multiple modalities (ex. taste and hearing). Here we had three aims: first, determine if cholinergic activation is involved in consumption behavior. Second, if so, distinguish the impact of the caloric value by using saccharin, a zero calorie sweetener. Lastly, we tested the perception of acoustic stimuli by measuring the acoustic startle reflex (ASR).Male Sprague‐Dawley rats were bilaterally cannulated into the PPTg, then housed in a contact lickometer and placed under food restriction lasting the entire consumption experiment (water ad lib.). The lickometer recorded each individual lick to give insight into the microstructure of the consumption behavior. Treatment consisted of a microinjection of either 1 μL of aCSF or 1 μL of 10 μM UII into the PPTg, and the rats were immediately given access to either sucrose or saccharin. For the remaining five days rats were allowed one hour access per day to the same sweet solution without any further treatments. ASR testing consisted of a baseline (no treatment), treatment day, and two additional days (no treatment). Immediately following the microinjection of UII, consumption of both saccharin and sucrose increased compared to controls. The significant difference in sucrose consumption lasted two days post‐treatment while saccharin consumption was significantly different for three days after treatment. The ASR was significantly increased the day after treatment in the rats treated with UII.Activating cholinergic PPTg neurons may lead to a miscalculation of the salience of external stimuli, implicating the importance of cholinergic input in modulating a variety of behaviors. This potential plastic change can give rise to further studies in the role of sensory processing on reward related‐behaviors at the level of the PPTg cholinergic neurons.Support or Funding InformationThe Howard T. Blane Director's Award in the Addictions (BDAA) from Research Institute on AddictionThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Target selectivity of septal cholinergic neurons in the medial and lateral entorhinal cortex

The entorhinal cortex (EC) plays a pivotal role in processing and conveying spatial information to the hippocampus. It has long been known that EC neurons are modulated by cholinergic input from the medial septum. However, little is known as to how synaptic release of acetylcholine affects the different cell types in EC. Here we combined optogenetics and patch-clamp recordings to study the effect of cholinergic axon stimulation on distinct neurons in EC. We found dense cholinergic innervations that terminate in layer I and II (LI and LII). Light-activated stimulation of septal cholinergic projections revealed differential responses in excitatory and inhibitory neurons in LI and LII of both medial and lateral EC. We observed depolarizing responses mediated by nicotinic and muscarinic receptors primarily in putative serotonin receptor (p5HT3R)-expressing interneurons. Hyperpolarizing muscarinic receptor-mediated responses were found predominantly in excitatory cells. Additionally, some excitatory as well as a higher fraction of inhibitory neurons received mono- and/or polysynaptic GABAergic inputs, revealing that medial septum cholinergic neurons have the capacity to corelease GABA alongside acetylcholine. Notably, the synaptic effects of acetylcholine were similar in neurons of both medial and lateral EC. Taken together, our findings demonstrate that EC activity may be differentially modulated via the activation or the suppression of distinct subsets of LI and LII neurons by the septal cholinergic system.

Cell type specific tracing of the subcortical input to primary visual cortex from the basal forebrain

The basal forebrain provides cholinergic inputs to primary visual cortex (V1) that play a key modulatory role on visual function. While basal forebrain afferents terminate in the infragranular layers of V1, acetylcholine is delivered to more superficial layers through volume transmission. Nevertheless, direct synaptic contact in deep layers 5 and 6 may provide a more immediate effect on V1 modulation. Using helper viruses with cell type specific promoters to target retrograde infection of pseudotyped and genetically modified rabies virus evidence was found for direct synaptic input onto V1 inhibitory neurons. These inputs were similar in number to geniculocortical inputs and, therefore, considered robust. In contrast, while clear evidence for dorsal lateral geniculate nucleus input to V1 excitatory neurons was found, there was no evidence of direct synaptic input from the basal forebrain. These results suggest a direct and more immediate influence of the basal forebrain on local V1 inhibition.

The pedunculopontine and laterodorsal tegmental nuclei in the kainate model of epilepsy

Prior studies showed that epilepsy can be associated with reorganization of the septohippocampal cholinergic fiber system. Using the kainate model of epilepsy, we wished to further examine the structural integrity of the mesopontine tegmental nuclei (pedunculopontine, PPN, and laterodorsal, LDT), which provide the cholinergic input to the thalamus. It was found that the total numbers of the PPN and LDT cells immunoreactive to the vesicular acetylcholine transporter did not differ between control and epileptic rats. However, the cholinergic cells had enlarged perikarya in epileptic rats. We further examined the effects of epilepsy on the distribution pattern of cholinergic fiber varicosities in the parafascicular nucleus, one of the principal thalamic targets of PPN projections. The density of cholinergic varicosities, represented by two distinct populations, was increased in epileptic rats. These data provide the first morphological evidence for structural alterations in mesopontine cholinergic neurons in experimental epilepsy. They suggest dysfunctional cholinergic transmission in the brainstem-thalamic pathway, which may partly account for various epilepsy-related neurological disturbances.