Cholinergic Axons Research Articles

Nicotinic Cholinergic System in the Hypothalamus Modulates the Activity of the Hypothalamic Neuropeptides During the Stress Response

BackgroundThe hypothalamus harbors high levels of cholinergic neurons and axon terminals. Nicotinic acetylcholine receptors, which play an important role in cholinergic neurotransmission, are expressed abundantly in the hypothalamus. Accumulating evidence reveals a regulatory role for nicotine in the regulation of the stress responses. The present review will discuss the hypothalamic neuropeptides and their interaction with the nicotinic cholinergic system. The anatomical distribution of the cholinergic neurons, axon terminals and nicotinic receptors in discrete hypothalamic nuclei will be described. The effect of nicotinic cholinergic neurotransmission and nicotine exposure on hypothalamic-pituitaryadrenal (HPA) axis regulation at the hypothalamic level will be analyzed in view of the different neuropeptides involved.MethodsPublished research related to nicotinic cholinergic regulation of the HPA axis activity at the hypothalamic level is reviewed.ResultsThe nicotinic cholinergic system is one of the major modulators of the HPA axis activity. There is substantial evidence supporting the regulation of hypothalamic neuropeptides by nicotinic acetylcholine receptors. However, most of the studies showing the nicotinic regulation of hypothalamic neuropeptides have employed systemic administration of nicotine. Additionally, we know little about the nicotinic receptor distribution on neuropeptide-synthesizing neurons in the hypothalamus and the physiological responses they trigger in these neurons.ConclusionDisturbed functioning of the HPA axis and hypothalamic neuropeptides results in pathologies such as depression, anxiety disorders and obesity, which are common and significant health problems. A better understanding of the nicotinic regulation of hypothalamic neuropeptides will aid in drug development and provide means to cope with these diseases. Considering that nicotine is also an abused substance, a better understanding of the role of the nicotinic cholinergic system on the HPA axis will aid in developing improved therapeutic strategies for smoking cessation.

Nicotinic Cholinergic Receptors in VTA Glutamate Neurons Modulate Excitatory Transmission

SUMMARYVentral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.

Autonomic reinnervation and functional regeneration in autologous transplanted submandibular glands in patients with severe keratoconjunctivitis sicca

Autologous submandibular gland (SMG) transplantation has been proved to ameliorate the discomforts in patients with severe keratoconjunctivitis sicca. The transplanted glands underwent a hypofunctional period and then restored secretion spontaneously. This study aims to investigate whether autonomic nerves reinnervate the grafts and contribute to the functional recovery, and further determine the origin of these nerves. Parts of the transplanted SMGs were collected from the epiphora patients, and a rabbit SMG transplantation model was established to fulfill the serial observation on the transplanted glands with time. The results showed that autonomic nerves distributed in the transplanted SMGs and parasympathetic ganglionic cells were observed in the stroma of the glands. Low-dense and unevenly distributed cholinergic axons, severe acinar atrophy and fibrosis were visible in the patients’ glands 4–6 months post-transplantation, whereas the cholinergic axon density and acinar area were increased with time. The acinar area or the secretory flow rate of the transplanted glands was statistically correlated with the cholinergic axon density in the rabbit model, respectively. Meanwhile, large cholinergic nerve trunks were found to locate in the temporal fascia lower to the gland, and sympathetic plexus concomitant with the arteries was observed both in the adjacent fascia and in the stroma of the glands. In summary, the transplanted SMGs are reinnervated by autonomic nerves and the cholinergic nerves play a role in the morphological and functional restoration of the glands. Moreover, these autonomic nerves might originate from the auriculotemporal nerve and the sympathetic plexus around the supplying arteries.

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.

Annals of Neurology: Volume 82, Number 3, September 2017

ON THE COVER: A nerve fascicle of a human thoracodorsal nerve stained by using immunofluorescence against choline acetyltransferase (green) to visualize cholinergic (motor) axons and neurofilament (red), which stains all myelinated and unmyelinated axons irrespective of their axon quality (motor or sensory). Doubly labeled motor axons appear yellow due to the overlap of green and red. Note the uneven distribution of motor axons, which constitute only about 10% of axons in spinal nerves that innervate the human upper limbs. See Gesslbauer et al, pp. 396–408 in this issue for further details. Ann Neurol 2017;82:1–1

Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization.

Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

Functional and structural microanatomy of the fetal sciatic nerve.

The ultrastructure of a nerve has implications for surgical nerve repair. The aim of our study was to characterize the fascicular versus fibrillar anatomy and the autonomic versus somatic nature of the fetal sciatic nerve (SN). Immunohistochemistry for vesicular acetylcholine transporter, tyrosine hydroxylase, and peripheral myelin protein 22 was performed to identify cholinergic, adrenergic, and somatic axons, respectively, in the human fetal SN. Two-dimensional (2D) analysis and 3D reconstructions were performed. The fetal SN is composed of one-third stromal tissue and two-thirds neural tissue. Autonomic fibers are predominant over somatic fibers within the neural tissue. The distribution of somatic fibers is initially random, but then become topographically organized after intra- and interfascicular rearrangements have occurred within the nerve. The fetal model presents limitations but enables illustration of the nature of the nerve fibers and the 3D fascicular anatomy of the SN. Muscle Nerve 56: 787-796, 2017.

Striatal Cholinergic Interneurons Modulate Spike-Timing in Striosomes and Matrix by an Amphetamine-Sensitive Mechanism.

The striatum is key for action-selection and the motivation to move. Dopamine and acetylcholine release sites are enriched in the striatum and are cross-regulated, possibly to achieve optimal behavior. Drugs of abuse, which promote abnormally high dopamine release, disrupt normal action-selection and drive restricted, repetitive behaviors (stereotypies). Stereotypies occur in a variety of disorders including obsessive-compulsive disorder, autism, schizophrenia and Huntington's disease, as well as in addictive states. The severity of drug-induced stereotypy is correlated with induction of c-Fos expression in striosomes, a striatal compartment that is related to the limbic system and that directly projects to dopamine-producing neurons of the substantia nigra. These characteristics of striosomes contrast with the properties of the extra-striosomal matrix, which has strong sensorimotor and associative circuit inputs and outputs. Disruption of acetylcholine signaling in the striatum blocks the striosome-predominant c-Fos expression pattern induced by drugs of abuse and alters drug-induced stereotypy. The activity of striatal cholinergic interneurons is associated with behaviors related to sensory cues, and cortical inputs to striosomes can bias action-selection in the face of conflicting cues. The neurons and neuropil of striosomes and matrix neurons have observably separate distributions, both at the input level in the striatum and at the output level in the substantia nigra. Notably, cholinergic axons readily cross compartment borders, providing a potential route for local cross-compartment communication to maintain a balance between striosomal and matrix activity. We show here, by slice electrophysiology in transgenic mice, that repetitive evoked firing patterns in striosomal and matrix striatal projection neurons (SPNs) are interrupted by optogenetic activation of cholinergic interneurons either by the addition or the deletion of spikes. We demonstrate that this cholinergic modulation of projection neurons is blocked in brain slices taken from mice exposed to amphetamine and engaged in amphetamine-induced stereotypy, and lacking responsiveness to salient cues. Our findings support a model whereby activity in striosomes is normally under strong regulation by cholinergic interneurons, favoring behavioral flexibility, but that in animals with drug-induced stereotypy, this cholinergic signaling breaks down, resulting in differential modulation of striosomal activity and an inability to bias action-selection according to relevant sensory cues.

The Input-Output Relationship of the Cholinergic Basal Forebrain

Basal forebrain cholinergic neurons influence cortical state, plasticity, learning, and attention. They collectively innervate the entire cerebral cortex, differentially controlling acetylcholine efflux across different cortical areas and timescales. Such control might be achieved by differential inputs driving separable cholinergic outputs, although no input-output relationship on a brain-wide level has ever been demonstrated. Here, we identify input neurons to cholinergic cells projecting to specific cortical regions by infecting cholinergic axon terminals withamonosynaptically restricted viral tracer. This approach revealed several circuit motifs, such as central amygdala neurons synapsing onto basolateral amygdala-projecting cholinergic neurons or strong somatosensory cortical input to motor cortex-projecting cholinergic neurons. The presence of input cells in the parasympathetic midbrain nuclei contacting frontally projecting cholinergic neurons suggest that the network regulating the inner eye muscles are additionally regulating cortical state via acetylcholine efflux. This dataset enables future circuit-level experiments to identify drivers of known cortical cholinergic functions.

Pupil fluctuations track rapid changes in adrenergic and cholinergic activity in cortex.

Rapid variations in cortical state during wakefulness have a strong influence on neural and behavioural responses and are tightly coupled to changes in pupil size across species. However, the physiological processes linking cortical state and pupil variations are largely unknown. Here we demonstrate that these rapid variations, during both quiet waking and locomotion, are highly correlated with fluctuations in the activity of corticopetal noradrenergic and cholinergic projections. Rapid dilations of the pupil are tightly associated with phasic activity in noradrenergic axons, whereas longer-lasting dilations of the pupil, such as during locomotion, are accompanied by sustained activity in cholinergic axons. Thus, the pupil can be used to sensitively track the activity in multiple neuromodulatory transmitter systems as they control the state of the waking brain.

Parallel processing by cortical inhibition enables context-dependent behavior.

Physical features of sensory stimuli are fixed, but sensory perception is context-dependent. The precise mechanisms that govern contextual modulation remain unknown. Here, we trained mice to switch between two contexts: passively listening to pure tones vs. performing a recognition task for the same stimuli. Two-photon imaging showed that many excitatory neurons in auditory cortex were suppressed, while some cells became more active during behavior. Whole-cell recordings showed that excitatory inputs were only modestly affected by context, but inhibition was more sensitive, with PV, SOM+, and VIP+ interneurons balancing inhibition/disinhibition within the network. Cholinergic modulation was involved in context-switching, with cholinergic axons increasing activity during behavior and directly depolarizing inhibitory cells. Network modeling captured these findings, but only when modulation coincidently drove all three interneuron subtypes, ruling out either inhibition or disinhibition alone as sole mechanism for active engagement. Parallel processing of cholinergic modulation by cortical interneurons therefore enables context-dependent behavior.

Cholinergic neurodegeneration in an Alzheimer mouse model overexpressing amyloid-precursor protein with the Swedish-Dutch-Iowa mutations

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder that is mainly characterized by beta-amyloid (Aβ) plaque deposition, Tau pathology and dysfunction of the cholinergic system causing memory impairment. The aim of the present study was to examine (1) anxiety and cognition, (2) Aβ plaque deposition and (3) degeneration of cholinergic neurons in the nucleus basalis of Meynert (nbM) and cortical cholinergic innervation in an Alzheimer mouse model (APP_SweDI; overexpressing amyloid precursor protein (APP) with the Swedish K670N/M671L, Dutch E693Q, and Iowa D694N mutations). Our results show that 12-month-old APP_SweDI mice were more anxious and had more memory impairment. A large number of Aβ plaques were already visible at the age of 6 months and increased with age. A significant decrease in cholinergic neurons was seen in the transgenic mouse model in comparison to the wild-type mice, identified by immunohistochemistry against choline acetyltransferase (ChAT) and p75 neurotrophin receptor as well as by in situ hybridization. Moreover, a significant decrease in cortical cholinergic fiber density was found in the transgenic mice as compared to the wild-type. In the cerebral cortex of APP_SweDI mice, swollen cholinergic varicosities were seen in the vicinity of Aβ plaques. In conclusion, the present study shows that in an AD mouse model (APP_SweDI mice) a high Aβ plaque load in the cortex causes damage to cholinergic axons in the cortex, followed by subsequent retrograde-induced cell death of cholinergic neurons and some forms of compensatory processes. This degeneration was accompanied by enhanced anxiety and impaired cognition.

Localization of organic cation transporter 2 (OCT2) in monoaminergic and cholinergic axon terminals of the mouse brain

Organic cation transporters (OCTs) are low-affinity, high-capacity carriers that mediate sodium-independent transport for biogenic cations, including catecholamine, serotonin, histamine, and choline/acetylcholine. Among them, OCT2 is expressed in neurons of the central nervous system. Although previous studies show OCT2 expression in several populations of cholinergic and monoaminergic neurons, the regional distribution of OCT2 in the brain remains largely unknown. Here we performed immunohistochemical analyses to reveal the distribution of OCT2 throughout the mouse forebrain. OCT2 immunoreactivity was widely distributed, with substantial regional specificity in cortical and subcortical structures including the hippocampus, striatum, and some subdivisions of the amygdala and extended amygdala. Interestingly, OCT2 appeared as punctate, bouton-like labeling in cholinergic, dopaminergic, and serotonergic axon terminals that were co-labeled with presynaptic neurochemical markers. We also co-labeled OCT2 and an anterograde tract-tracer injected into the locus coeruleus, demonstrating that OCT2 was localized to presumptive noradrenergic terminals in the forebrain. Together, our results demonstrated that the polyspecific cation transporter OCT2 is distributed in cholinergic and monoaminergic terminals in various forebrain regions, suggesting that OCT2 could play a role in regulating presynaptic reuptake and recycling of choline and monoamines.

Structural basis for cholinergic regulation of neural circuits in the mouse olfactory bulb.

Odor information is regulated by olfactory inputs, bulbar interneurons, and centrifugal inputs in the olfactory bulb (OB). Cholinergic neurons projecting from the nucleus of the horizontal limb of the diagonal band of Broca and the magnocellular preoptic nucleus are one of the primary centrifugal inputs to the OB. In this study, we focused on cholinergic regulation of the OB and analyzed neural morphology with a particular emphasis on the projection pathways of cholinergic neurons. Single-cell imaging of a specific neuron within dense fibers is critical to evaluate the structure and function of the neural circuits. We labeled cholinergic neurons by infection with virus vector and then reconstructed them three-dimensionally. We also examined the ultramicrostructure of synapses by electron microscopy tomography. To further clarify the function of cholinergic neurons, we performed confocal laser scanning microscopy to investigate whether other neurotransmitters are present within cholinergic axons in the OB. Our results showed the first visualization of complete cholinergic neurons, including axons projecting to the OB, and also revealed frequent axonal branching within the OB where it innervated multiple glomeruli in different areas. Furthermore, electron tomography demonstrated that cholinergic axons formed asymmetrical synapses with a morphological variety of thicknesses of the postsynaptic density. Although we have not yet detected the presence of other neurotransmitters, the range of synaptic morphology suggests multiple modes of transmission. The present study elucidates the ways that cholinergic neurons could contribute to the elaborate mechanisms involved in olfactory processing in the OB. J. Comp. Neurol. 525:574-591, 2017. © 2016 Wiley Periodicals, Inc.

Segregated cholinergic transmission modulates dopamine neurons integrated in distinct functional circuits.

Dopamine neurons in the ventral tegmental area (VTA) receive cholinergic innervation from brainstem structures that are associated with either movement or reward. Whereas cholinergic neurons of the pedunculopontine nucleus (PPN) carry an associative/motor signal, those of the laterodorsal tegmental nucleus (LDT) convey limbic information. We used optogenetics and in vivo juxtacellular recording and labeling to examine the influence of brainstem cholinergic innervation of distinct neuronal subpopulations in the VTA. We found that LDT cholinergic axons selectively enhanced the bursting activity of mesolimbic dopamine neurons that were excited by aversive stimulation. In contrast, PPN cholinergic axons activated and changed the discharge properties of VTA neurons that were integrated in distinct functional circuits and were inhibited by aversive stimulation. Although both structures conveyed a reinforcing signal, they had opposite roles in locomotion. Our results demonstrate that two modes of cholinergic transmission operate in the VTA and segregate the neurons involved in different reward circuits.

The Basal Forebrain and Motor Cortex Provide Convergent yet Distinct Movement-Related Inputs to the Auditory Cortex

Cholinergic inputs to the auditory cortex from the basal forebrain (BF) are important to auditory processing and plasticity, but little is known about the organization of these synapses onto different auditory cortical neuron types, how they influence auditory responsiveness, and their activity patterns during various behaviors. Using intersectional tracing, optogenetic circuit mapping, and invivo calcium imaging, we found that cholinergic axons arising from the caudal BF target major excitatory and inhibitory auditory cortical cell types, rapidly modulate auditory cortical tuning, and display fast movement-related activity. Furthermore, the BF and the motor cortex-another source of movement-related activity-provide convergent input onto some of the same auditory cortical neurons. Cholinergic and motor cortical afferents to the auditory cortex display distinct activity patterns and presynaptic partners, indicating that the auditory cortex integrates bottom-up cholinergic signals related to ongoing movements and arousal with top-down information concerning impending movements and motor planning.

Localization of the M2 muscarinic cholinergic receptor in dendrites, cholinergic terminals, and noncholinergic terminals in the rat basolateral amygdala: An ultrastructural analysis.

Activation of M2 muscarinic receptors (M2Rs) in the rat anterior basolateral nucleus (BLa) is critical for the consolidation of memories of emotionally arousing events. The present investigation used immunocytochemistry at the electron microscopic level to determine which structures in the BLa express M2Rs. In addition, dual localization of M2R and the vesicular acetylcholine transporter protein (VAChT), a marker for cholinergic axons, was performed to determine whether M2R is an autoreceptor in cholinergic axons innervating the BLa. M2R immunoreactivity (M2R-ir) was absent from the perikarya of pyramidal neurons, with the exception of the Golgi complex, but was dense in the proximal dendrites and axon initial segments emanating from these neurons. Most perikarya of nonpyramidal neurons were also M2R-negative. About 95% of dendritic shafts and 60% of dendritic spines were M2 immunoreactive (M2R(+) ). Some M2R(+) dendrites had spines, suggesting that they belonged to pyramidal cells, whereas others had morphological features typical of nonpyramidal neurons. M2R-ir was also seen in axon terminals, most of which formed asymmetrical synapses. The main targets of M2R(+) terminals forming asymmetrical (putative excitatory) synapses were dendritic spines, most of which were M2R(+) . The main targets of M2R(+) terminals forming symmetrical (putative inhibitory or neuromodulatory) synapses were unlabeled perikarya and M2R(+) dendritic shafts. M2R-ir was also seen in VAChT(+) cholinergic terminals, indicating a possible autoreceptor role. These findings suggest that M2R-mediated mechanisms in the BLa are very complex, involving postsynaptic effects in dendrites as well as regulating release of glutamate, γ-aminobutyric acid, and acetylcholine from presynaptic axon terminals. J. Comp. Neurol. 524:2400-2417, 2016. © 2016 Wiley Periodicals, Inc.

Extrinsic Sources of Cholinergic Innervation of the Striatal Complex: A Whole-Brain Mapping Analysis.

Acetylcholine in the striatal complex plays an important role in normal behavior and is affected in a number of neurological disorders. Although early studies suggested that acetylcholine in the striatum (STR) is derived almost exclusively from cholinergic interneurons (CIN), recent axonal mapping studies using conditional anterograde tracing have revealed the existence of a prominent direct cholinergic pathway from the pedunculopontine and laterodorsal tegmental nuclei to the dorsal striatum and nucleus accumbens. The identification of the importance of this pathway is essential for creating a complete model of cholinergic modulation in the striatum, and it opens the question as to whether other populations of cholinergic neurons may also contribute to such modulation. Here, using novel viral tracing technologies based on phenotype-specific fluorescent reporter expression in combination with retrograde tracing, we aimed to define other sources of cholinergic innervation of the striatum. Systematic mapping of the projections of all cholinergic structures in the brain (Ch1 to Ch8) by means of conditional tracing of cholinergic axons, revealed that the only extrinsic source of cholinergic innervation arises in the brainstem pedunculopontine and laterodorsal tegmental nuclei. Our results thus place the pedunculopontine and laterodorsal nuclei in a key and exclusive position to provide extrinsic cholinergic modulation of the activity of the striatal systems.

Intrinsic and extrinsic innervation of the heart in zebrafish (Danio rerio).

In the vertebrate heart the intracardiac nervous system is the final common pathway for autonomic control of cardiac output, but the neuroanatomy of this system is not well understood. In this study we investigated the innervation of the heart in a model vertebrate, the zebrafish. We used antibodies against acetylated tubulin, human neuronal protein C/D, choline acetyltransferase, tyrosine hydroxylase, neuronal nitric oxide synthase, and vasoactive intestinal polypeptide to visualize neural elements and their neurotransmitter content. Most neurons were located at the venous pole in a plexus around the sinoatrial valve; mean total number of cells was 197 ± 23, and 92% were choline acetyltransferase positive, implying a cholinergic role. The plexus contained cholinergic, adrenergic, and nitrergic axons and vasoactive intestinal polypeptide-positive terminals, some innervating somata. Putative pacemaker cells near the plexus showed immunoreactivity for hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and the transcription factor Islet-1 (Isl1). The neurotracer neurobiotin showed that extrinsic axons from the left and right vagosympathetic trunks innervated the sinoatrial plexus proximal to their entry into the heart; some extrinsic axons from each trunk also projected into the medial dorsal plexus region. Extrinsic axons also innervated the atrial and ventricular walls. An extracardiac nerve trunk innervated the bulbus arteriosus and entered the arterial pole of the heart to innervate the proximal ventricle. We have shown that the intracardiac nervous system in the zebrafish is anatomically and neurochemically complex, providing a substrate for autonomic control of cardiac effectors in all chambers.

Synaptic connectivity of the cholinergic axons in the olfactory bulb of the cynomolgus monkey.

The olfactory bulb (OB) of mammals receives cholinergic afferents from the horizontal limb of the diagonal band of Broca (HDB). At present, the synaptic connectivity of the cholinergic axons on the circuits of the OB has only been investigated in the rat. In this report, we analyze the synaptic connectivity of the cholinergic axons in the OB of the cynomolgus monkey (Macaca fascicularis). Our aim is to investigate whether the cholinergic innervation of the bulbar circuits is phylogenetically conserved between macrosmatic and microsmatic mammals. Our results demonstrate that the cholinergic axons form synaptic contacts on interneurons. In the glomerular layer, their main targets are the periglomerular cells, which receive axo-somatic and axo-dendritic synapses. In the inframitral region, their main targets are the granule cells, which receive synaptic contacts on their dendritic shafts and spines. Although the cholinergic boutons were frequently found in close vicinity of the dendrites of principal cells, we have not found synaptic contacts on them. From a comparative perspective, our data indicate that the synaptic connectivity of the cholinergic circuits is highly preserved in the OB of macrosmatic and microsmatic mammals.