Retrograde Axonal Transport of Neurotrophins in Basal Forebrain Cholinergic Neurons.

Axonal transport is key for the survival and function of all neurons. This process is especially important in basal forebrain cholinergic neurons due to their extremely long and diffuse axonal projections. These neurons are critical for learning and memory and degenerate rapidly in age-related neurodegenerative disorders like Alzheimer's and Parkinson's disease. The vulnerability of these neurons to age-related neurodegeneration may be partially attributed to their reliance on retrograde axonal transport for neurotrophic support. Unfortunately, little is known about the molecular biology underlying the retrograde transport dynamics of these neurons due to the difficulty associated with their maintenance in vitro. Here, we outline a protocol for culturing primary rodent basal forebrain cholinergic neurons in microfluidic chambers, devices designed specifically for the study of axonal transport in vitro. We outline protocols for labeling neurotrophins and tracking neurotrophin transport in these neurons. Our protocols can also be used to study axonal transport in other types of primary neurons such as cortical and hippocampal neurons.

Quantitative EEG and cholinergic basal forebrain atrophy in Parkinson's disease and mild cognitive impairment.

Cholinergic degeneration is a key feature of dementia in neurodegenerative conditions including Alzheimer's disease (AD) and Parkinson's disease (PD). Quantitative electro-encephalography (EEG) metrics are altered in both conditions from early stages, and recent research in people with Lewy body and AD dementia suggests these changes may be associated with atrophy in cholinergic basal forebrain nuclei (cBF). To determine if these relationships exist in predementia stages of neurodegenerative conditions, we studied resting-state EEG and in vivo cBF volumes in 31 people with PD (without dementia), 21 people with mild cognitive impairment (MCI), and 21 age-matched controls. People with PD showed increased power in slower frequencies and reduced alpha reactivity compared to controls. Volumes of cholinergic cell clusters corresponding to the medial septum and vertical and horizontal limb of the diagonal band, and the posterior nucleus basalis of Meynert, correlated positively with; alpha reactivity in people with PD (p< 0.01); and pre-alpha power in people with MCI (p< 0.05). These results suggest that alpha reactivity and pre-alpha power are related to changes in cBF volumes in MCI and PD without dementia.

The cryptic gonadotropin-releasing hormone neuronal system of human basal ganglia.

Human reproduction is controlled by ~2000 hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Here, we report the discovery and characterization of additional ~150,000-200,000 GnRH-synthesizing cells in the human basal ganglia and basal forebrain. Nearly all extrahypothalamic GnRH neurons expressed the cholinergic marker enzyme choline acetyltransferase. Similarly, hypothalamic GnRH neurons were also cholinergic both in embryonic and adult human brains. Whole-transcriptome analysis of cholinergic interneurons and medium spiny projection neurons laser-microdissected from the human putamen showed selective expression of GNRH1 and GNRHR1 autoreceptors in the cholinergic cell population and uncovered the detailed transcriptome profile and molecular connectome of these two cell types. Higher-order non-reproductive functions regulated by GnRH under physiological conditions in the human basal ganglia and basal forebrain require clarification. The role and changes of GnRH/GnRHR1 signaling in neurodegenerative disorders affecting cholinergic neurocircuitries, including Parkinson's and Alzheimer's diseases, need to be explored.

Age-associated decline in septum neuronal activation during spatial learning in homing pigeons (Columba livia).

The relationship between hippocampal aging and spatial-cognitive decline in birds has recently been investigated. However, like its mammalian counterpart, the avian hippocampus does not work in isolation and its relationship to the septum is of particular interest. The current study aimed to investigate the effects of age on septum (medial and lateral) and associated nucleus of the diagonal band (NDB) neuronal activation (as indicated by c-Fos expression) during learning of a spatial, delayed non-match-to-sample task conducted in a modified radial arm maze. The results indicated significantly reduced septum, but not NDB, activation during spatial learning in older pigeons. We also preliminarily investigated the effect of age on the number of cholinergic septum and NDB neurons (as indicated by expression of choline acetyltransferase; ChAT). Although underpowered to reveal a statistical effect, the data suggest that older pigeons have substantially fewer ChAT-expressing cells in the septum compared to younger pigeons. The data support the hypothesis that reduced activation of the septum contributes to the age-related, spatial cognitive impairment in pigeons.

Regulation of sleep homeostasis mediator adenosine by basal forebrain glutamatergic neurons.

Sleep and wakefulness are homeostatically regulated by a variety of factors, including adenosine. However, how neural activity underlying the sleep-wake cycle controls adenosine release in the brain remains unclear. Using a newly developed genetically encoded adenosine sensor, we found an activity-dependent rapid increase in the concentration of extracellular adenosine in mouse basal forebrain (BF), a critical region controlling sleep and wakefulness. Although the activity of both BF cholinergic and glutamatergic neurons correlated with changes in the concentration of adenosine, optogenetic activation of these neurons at physiological firing frequencies showed that glutamatergic neurons contributed much more to the adenosine increase. Mice with selective ablation of BF glutamatergic neurons exhibited a reduced adenosine increase and impaired sleep homeostasis regulation. Thus, cell type-specific neural activity in the BF dynamically controls sleep homeostasis.

High-density EEG of auditory steady-state responses during stimulation of basal forebrain parvalbumin neurons.

We present high-density EEG datasets of auditory steady-state responses (ASSRs) recorded from the cortex of freely moving mice with or without optogenetic stimulation of basal forebrain parvalbumin (BF-PV) neurons, known as a subcortical hub circuit for the global workspace. The dataset of ASSRs without BF-PV stimulation (dataset 1) contains raw 36-channel EEG epochs of ASSRs elicited by 10, 20, 30, 40, and 50 Hz click trains and time stamps of stimulations. The dataset of ASSRs with BF-PV stimulation (dataset 2) contains raw 36-channel EEG epochs of 40-Hz ASSRs during BF-PV stimulation with latencies of 0, 6.25, 12.5, and 18.75 ms and time stamps of stimulations. We provide the datasets and step-by-step tutorial analysis scripts written in Python, allowing for descriptions of the event-related potentials, spectrograms, and the topography of power. We complement this experimental dataset with simulation results using a time-dependent perturbation on coupled oscillators. This publicly available dataset will be beneficial to the experimental and computational neuroscientists.

Neural Circuits for Sleep-Wake Regulation.

The neural mechanisms of sleep, a fundamental biological behavior from invertebrates to humans, have been a long-standing mystery and present an enormous challenge. Gradually, perspectives on the neurobiology of sleep have been more various with the technical innovations over the recent decades, and studies have now identified many specific neural circuits that selectively regulate the initiation and maintenance of wake, rapid eye movement (REM) sleep, and non-REM (NREM) sleep. The cholinergic system in basal forebrain (BF) that fire maximally during waking and REM sleep is one of the key neuromodulation systems related to waking and REM sleep. Here we outline the recent progress of the BF cholinergic system in sleep-wake cycle. The intricate local connectivity and multiple projections to other cortical and subcortical regions of the BF cholinergic system elaborately presented here form a conceptual framework for understanding the coordinating effects with the dissecting regions. This framework also provides evidences regarding the relationships between the general anesthesia and wakefulness/sleep cycle focusing on the neural circuitry of unconsciousness induced by anesthetic drugs.

Tumor Necrosis Factor α Influences Phenotypic Plasticity and Promotes Epigenetic Changes in Human Basal Forebrain Cholinergic Neuroblasts.

TNFα is the main proinflammatory cytokine implicated in the pathogenesis of neurodegenerative disorders, but it also modulates physiological functions in both the developing and adult brain. In this study, we investigated a potential direct role of TNFα in determining phenotypic changes of a recently established cellular model of human basal forebrain cholinergic neuroblasts isolated from the nucleus basalis of Meynert (hfNBMs). Exposing hfNBMs to TNFα reduced the expression of immature markers, such as nestin and ß-tubulin III, and inhibited primary cilium formation. On the contrary, TNFα increased the expression of TNFα receptor TNFR2 and the mature neuron marker MAP2, also promoting neurite elongation. Moreover, TNFα affected nerve growth factor receptor expression. We also found that TNFα induced the expression of DNA-methylation enzymes and, accordingly, downregulated genes involved in neuronal development through epigenetic mechanisms, as demonstrated by methylome analysis. In summary, TNFα showed a dual role on hfNBMs phenotypic plasticity, exerting a negative influence on neurogenesis despite a positive effect on differentiation, through mechanisms that remain to be elucidated. Our results help to clarify the complexity of TNFα effects in human neurons and suggest that manipulation of TNFα signaling could provide a potential therapeutic approach against neurodegenerative disorders.

Stress-Related Neuronal Clusters in Sublenticular Extended Amygdala of Basal Forebrain Show Individual Differences of Positions.

To understand functional neuronal circuits for emotion in the basal forebrain, patterns of neuronal activation were examined in mice by immunohistochemistry of immediate-early gene products (Zif268/Egr1 and c-Fos). In all mice examined, clusters of 30-50 neurons expressing Zif268 were found on both sides in the area between the extended amygdala (EA) and globus pallidus (GP), generally designated as sublenticular extended amygdala (SLEA). The clusters consisted of 79.9 ± 3.0% of GABAergic neurons in GAD65-mCherry mice. The expression of the cholinergic marker choline acetyltransferase and the GP markers parvalbumin, proenkephalin, and FoxP2 indicated that these neurons were different from known types of neurons in the EA and GP; therefore, we named them the sublenticular extended amygdalar Zif268/Egr1-expressing neuronal cluster (SLEA-zNC). Sublenticular extended amygdalar Zif268/Egr1-expressing neuronal clusters participated in stress processing because increasing numbers of cells were observed in SLEA-zNCs after exposure to restraint stress (RS), the induction of which was suppressed by diazepam treatment. Mapping SLEA-zNCs showed that their positions and arrangement varied individually; SLEA-zNCs were distributed asymmetrically and tended to be situated mainly in the middle region between the anterior commissure (AC) and posterior end of the GP. However, the total cell number in SLEA-zNCs was compatible between the right and left hemispheres after activation by RS. Therefore, SLEA-zNCs were distributed asymmetrically but were not lateralized. Because time courses of activation differed between the Zif268 and c-Fos, the sequential dual treatment of RSs enabled us to differentiate SLEA-zNCs activated by the first and second RS. The results supported that the same SLEA-zNCs responded to both the first and second RS, and this also applied for all SLEA-zNCs. Thus, we concluded that the cluster positions were invariable under RS in each mouse but were distributed differently between individual mice. We name these newly identified neuronal clusters as stress-related neuronal clusters, SLEA-zNCs, which are considered to be novel functional units of "islands of activation." Moreover, SLEA-zNCs were situated at different positions in all mice examined, showing individual differences in their positions.

Opposing Contributions of GABAergic and Glutamatergic Ventral Pallidal Neurons to Motivational Behaviors.

The ventral pallidum (VP) is critical for invigorating reward seeking and is also involved in punishment avoidance, but how it contributes to such opposing behavioral actions remains unclear. Here, we show that GABAergic and glutamatergic VP neurons selectively control behavior in opposing motivational contexts. In vivo recording combined with optogenetics in mice revealed that these two populations oppositely encode positive and negative motivational value, are differentially modulated by animal's internal state, and determine the behavioral response during motivational conflict. Furthermore, GABAergic VP neurons are essential for movements toward reward in a positive motivational context but suppress movements in an aversive context. In contrast, glutamatergic VP neurons are essential for movements to avoid a threat but suppress movements in an appetitive context. Our results indicate that GABAergic and glutamatergic VP neurons encode the drive for approach and avoidance, respectively, with the balance between their activities determining the type of motivational behavior.

Cocaine Dysregulates Dynorphin Modulation of Inhibitory Neurotransmission in the Ventral Pallidum in a Cell-Type-Specific Manner.

Cocaine-driven changes in the modulation of neurotransmission by neuromodulators are poorly understood. The ventral pallidum (VP) is a key structure in the reward system, in which GABA neurotransmission is regulated by opioid neuropeptides, including dynorphin. However, it is not known whether dynorphin acts differently on different cell types in the VP and whether its effects are altered by withdrawal from cocaine. Here, we trained wild-type, D1-Cre, A2A-Cre, or vGluT2-Cre:Ai9 male and female mice in a cocaine conditioned place preference protocol followed by 2 weeks of abstinence, and then recorded GABAergic synaptic input evoked either electrically or optogenetically onto identified VP neurons before and after applying dynorphin. We found that after cocaine CPP and abstinence dynorphin attenuated inhibitory input to VPGABA neurons through a postsynaptic mechanism. This effect was absent in saline mice. Furthermore, this effect was seen specifically on the inputs from nucleus accumbens medium spiny neurons expressing either the D1 or the D2 dopamine receptor. Unlike its effect on VPGABA neurons, dynorphin surprisingly potentiated the inhibitory input on VPvGluT2 neurons, but this effect was abolished after cocaine CPP and abstinence. Thus, dynorphin has contrasting influences on GABA input to VPGABA and VPvGluT2 neurons and these influences are affected differentially by cocaine CPP and abstinence. Collectively, our data suggest a role for dynorphin in withdrawal through its actions in the VP. As VPGABA and VPvGluT2 neurons have contrasting effects on drug-seeking behavior, our data may indicate a complex role for dynorphin in withdrawal from cocaine.SIGNIFICANCE STATEMENT The ventral pallidum consists mainly of GABAergic reward-promoting neurons, but it also encloses a subgroup of aversion-promoting glutamatergic neurons. Dynorphin, an opioid neuropeptide abundant in the ventral pallidum, shows differential modulation of GABA input to GABAergic and glutamatergic pallidal neurons and may therefore affect both the rewarding and aversive aspects of withdrawal. Indeed, abstinence after repeated exposure to cocaine alters dynorphin actions in a cell-type-specific manner; after abstinence dynorphin suppresses the inhibitory drive on the "rewarding" GABAergic neurons but ceases to modulate the inhibitory drive on the "aversive" glutamatergic neurons. This reflects a complex role for dynorphin in cocaine reward and abstinence.

Projection-Specific Potentiation of Ventral Pallidal Glutamatergic Outputs after Abstinence from Cocaine.

The ventral pallidum (VP) is a central node in the reward system that is strongly implicated in reward and addiction. Although the majority of VP neurons are GABAergic and encode reward, recent studies revealed a novel glutamatergic neuronal population in the VP [VP neurons expressing the vesicular glutamate transporter 2 (VPVGluT2)], whose activation generates aversion. Withdrawal from drugs has been shown to induce drastic synaptic changes in neuronal populations associated with reward, such as the ventral tegmental area (VTA) or nucleus accumbens neurons, but less is known about cocaine-induced synaptic changes in neurons classically linked with aversion. Here, we demonstrate that VPVGluT2 neurons contact different targets with different intensities, and that cocaine conditioned place preference (CPP) training followed by abstinence selectively potentiates their synapses on targets that encode aversion. Using whole-cell patch-clamp recordings combined with optogenetics in male and female transgenic mice, we show that VPVGluT2 neurons preferentially contact aversion-related neurons, such as lateral habenula neurons and VTA GABAergic neurons, with minor input to reward-related neurons, such as VTA dopamine and VP GABA neurons. Moreover, after cocaine CPP and abstinence, the VPVGluT2 input to the aversion-related structures is potentiated, whereas the input to the reward-related structures is depressed. Thus, cocaine CPP followed by abstinence may allow VPVGluT2 neurons to recruit aversion-related targets more readily and therefore be part of the mechanism underlying the aversive symptoms seen after withdrawal.SIGNIFICANCE STATEMENT The biggest problem in drug addiction is the high propensity to relapse. One central driver for relapse events is the negative aversive symptoms experienced by addicts during withdrawal. In this work, we propose a possible mechanism for the intensification of aversive feelings after withdrawal that involves the glutamatergic neurons of the ventral pallidum. We show not only that these neurons are most strongly connected to aversive targets, such as the lateral habenula, but also that, after abstinence, their synapses on aversive targets are strengthened, whereas the synapses on other rewarding targets are weakened. These data illustrate how after abstinence from cocaine, aversive pathways change in a manner that may contribute to relapse.

The Cholinergic Basal Forebrain Links Auditory Stimuli with Delayed Reinforcement to Support Learning.

Animals learn to fear conditioned sound stimuli (CSs) that accompany aversive unconditioned stimuli (USs). Auditory cortex (ACx) circuits reorganize to support auditory fear learning when CS-evoked activity temporally overlaps with US-evoked acetylcholine release from the basal forebrain. Here we describe robust fear learning and acetylcholine-dependent ACx plasticity even when the US is delayed by several seconds following CS offset. A 5-s CS-US gap was not bridged by persistent CS-evoked spiking throughout the trace period. Instead, within minutes following the start of conditioning, optogenetically identified basal forebrain neurons that encode the aversive US scaled up responses to the CS and increased functional coupling with the ACx. Over several days of conditioning, bulk imaging of cholinergic basal forebrain neurons revealed sustained sound-evoked activity that filled in the 5-s silent gap preceding the US. These findings identify a plasticity in the basal forebrain that supports learned associations between sensory stimuli and delayed reinforcement.

Basal forebrain somatostatin cells differentially regulate local gamma oscillations and functionally segregate motor and cognitive circuits.

The basal forebrain delivers extensive axonal projections to the cortical mantle regulating brain states and cognitive processing. Recent evidence has established the basal forebrain as a subcortical node of the default mode network that directionally influences cortical dynamics trough gamma oscillations, yet their synaptic origin has not been established. Here, we used optogenetic stimulation and in vivo recordings of transgenic mice to show that somatostatin neurons exert an anatomically specialized role in the coordination of subcortical gamma oscillations of the rostral basal forebrain. Indeed, the spike timing of somatostatin cells was tightly correlated with gamma oscillations in the ventral pallidum, but not in the medial septum. Consequently, optogenetic inactivation of somatostatin neurons selectively disrupted the amplitude and coupling of gamma oscillations only in the ventral pallidum. Moreover, photosupression of somatostatin cells produced specific behavioral interferences, with the ventral pallidum regulating locomotor speed and the medial septum modulating spatial working memory. Altogether, these data suggest that basal forebrain somatostatin cells can selectively synchronize local neuronal networks in the gamma band directly impinging on cortical dynamics and behavioral performance. This further supports the role of the basal forebrain as a subcortical switch commanding transitions between internally and externally oriented brain states.

Ventral Pallidum Is the Primary Target for Accumbens D1 Projections Driving Cocaine Seeking.

Outputs from the nucleus accumbens (NAc) include projections to the ventral pallidum and the ventral tegmental area and subtantia nigra in the ventral mesencephalon. The medium spiny neurons (MSN) that give rise to these pathways are GABAergic and consist of two populations of equal number that are segregated by differentially expressed proteins, including D1- and D2-dopamine receptors. Afferents to the ventral pallidum arise from both D1- and D2-MSNs, whereas the ventral mesencephalon is selectively innervated by D1-MSN. To determine the extent of collateralization of D1-MSN to these axon terminal fields we used retrograde labeling in transgenic mice expressing tdTomato selectively in D1-MSN, and found that a large majority of D1-MSN in either the shell or core subcompartments of the accumbens collateralized to both output structures. Approximately 70% of D1-MSNs projecting to the ventral pallidum collateralized to the ventral mesencephalon, whereas >90% of mesencephalic D1-MSN afferents collateralized to the ventral pallidum. In contrast, <10% of dorsal striatal D1-MSNs collateralized to both the globus pallidus and ventral mesencephalon. D1-MSN activation is required for conditioned cues to induce cocaine seeking. To determine which D1-MSN projection mediates cued cocaine seeking, we selectively transfected D1-MSNs in transgenic rats with an inhibitory Gi-coupled DREADD. Activation of the transfected Gi-DREADD with clozapine-N-oxide administered into the ventral pallidum, but not into the ventral mesencephalon, blocked cue-induced cocaine seeking. These data show that, although accumbens D1-MSNs largely collateralize to both the ventral pallidum and ventral mesencephalon, only D1-MSN innervation of the ventral pallidum is necessary for cue-induced cocaine seeking.SIGNIFICANCE STATEMENT Activity in D1 dopamine receptor-expressing neurons in the NAc is required for rodents to respond to cocaine-conditioned cues and relapse to drug seeking behaviors. The D1-expressing neurons project to both the ventral pallidum and ventral mesencephalon, and we found that a majority of the neurons that innervate the ventral pallidum also collateralize to the ventral mesencephalon. However, despite innervating both structures, only D1 innervation of the ventral pallidum mediates cue-induced cocaine seeking.

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.

Basal Forebrain Gating by Somatostatin Neurons Drives Prefrontal Cortical Activity.

The basal forebrain provides modulatory input to the cortex regulating brain states and cognitive processing. Somatostatin-expressing neurons constitute a heterogeneous GABAergic population known to functionally inhibit basal forebrain cortically projecting cells thus favoring sleep and cortical synchronization. However, it remains unclear if somatostatin cells can regulate population activity patterns in the basal forebrain and modulate cortical dynamics. Here, we demonstrate that somatostatin neurons regulate the corticopetal synaptic output of the basal forebrain impinging on cortical activity and behavior. Optogenetic inactivation of somatostatin neurons in vivo rapidly modified neural activity in the basal forebrain, with the consequent enhancement and desynchronization of activity in the prefrontal cortex, reflected in both neuronal spiking and network oscillations. Cortical activation was partially dependent on cholinergic transmission, suppressing slow waves and potentiating gamma oscillations. In addition, recruitment dynamics was cell type-specific, with interneurons showing similar temporal profiles, but stronger responses than pyramidal cells. Finally, optogenetic stimulation of quiescent animals during resting periods prompted locomotor activity, suggesting generalized cortical activation and increased arousal. Altogether, we provide physiological and behavioral evidence indicating that somatostatin neurons are pivotal in gating the synaptic output of the basal forebrain, thus indirectly controlling cortical operations via both cholinergic and non-cholinergic mechanisms.

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.

Different Subgroups of Cholinergic Neurons in the Basal Forebrain Are Distinctly Innervated by the Olfactory Regions and Activated Differentially in Olfactory Memory Retrieval.

The mammalian basal forebrain (BF), a heterogenous structure providing the primary cholinergic inputs to cortical and limbic structures, plays a crucial role in various physiological processes such as learning/memory and attention. Despite the involvement of the BF cholinergic neurons (BFCNs) in olfaction related memory has been reported, the underlying neural circuits remain poorly understood. Here, we combined viral trans-synaptic tracing systems and ChAT-cre transgenic mice to systematically reveal the relationship between the olfactory system and the different subsets of BFCNs. The retrograde adeno-associated virus and rabies virus (AAV-RV) tracing showed that different subregional BFCNs received diverse inputs from multiple olfactory cortices. The cholinergic neurons in medial and caudal horizontal diagonal band Broca (HDB), magnocellular preoptic area (MCPO) and ventral substantia innominate (SI; hereafter HMS complex, HMSc) received the inputs from the entire olfactory system such as the olfactory bulb (OB), anterior olfactory nucleus (AON), entorhinal cortex (ENT), basolateral amygdala and especially the piriform cortex (PC) and hippocampus (HIP); while medial septum (MS/DB) and a part of rostral HDB (hereafter MS/DB complex, MS/DBc), predominantly from HIP; and nucleus basalis Meynert (NBM) and dorsal SI (hereafter NBM complex, NBMc), mainly from the central amygdala. The anterograde vesicular stomatitis virus (VSV) tracing further validated that the major target of the OB to the BF is HMSc. To correlate these structural relations between the BFCNs and olfactory functions, the neurons activated in the BF during olfaction related task were mapped with c-fos immunostaining. It was found that some of the BFCNs were activated in go/no-go olfactory discrimination task, but with different activated patterns. Interestingly, the BFCNs in HMSc were more significantly activated than the other subregions. Therefore, our data have demonstrated that among the different subgroups of BFCNs, HMSc is more closely related to the olfactory system, both structurally and functionally. This work provides the evidence for distinct roles of different subsets of BFNCs in olfaction associated memory.

Cholinergic basal forebrain neurons regulate fear extinction consolidation through p75 neurotrophin receptor signaling.

Cholinergic basal forebrain (cBF)-derived neurotransmission plays a crucial role in regulating neuronal function throughout the cortex, yet the mechanisms controlling cholinergic innervation to downstream targets have not been elucidated. Here we report that removing the p75 neurotrophin receptor (p75NTR) from cBF neurons induces a significant impairment in fear extinction consolidation. We demonstrate that this is achieved through alterations in synaptic connectivity and functional activity within the medial prefrontal cortex. These deficits revert back to wild-type levels upon re-expression of the active domain of p75NTR in adult animals. These findings demonstrate a novel role for cholinergic neurons in fear extinction consolidation and suggest that neurotrophic signaling is a key regulator of cholinergic-cortical innervation and function.