Plasma cholinesterase activity: A benchmark for rapid detection of pesticide poisoning in an avian scavenger.

Poisoning due to exposure to organophosphate and carbamate pesticides is a common threat for many wildlife species, especially for scavengers such as vultures. The Griffon vulture population (Gyps fulvus), for instance, is deteriorating in the Eastern Mediterranean, and is considered to be critically endangered in Israel, where 48 out of 107 (45 %) known injury/mortality cases in 2010-2021 were caused by poisoning. Lack of specific clinical indications, together with levels of organophosphate or carbamate pesticides too low to detect, challenge the ability to diagnose and treat such poisoning events. The activity of cholinesterase (ChE) in plasma has the potential to serve as an effective biomarker for monitoring exposure to anticholinesterase pesticides in live vultures. Yet, the applicability of this approach has been limited by intra- and inter-species variations in ChE basal levels. The present study aims to provide a benchmark for ChE activity levels in healthy Griffons and their intra-species variation. Blood samples from free-roaming (n = 231) and captive (n = 63) Griffons were collected during routine monitoring, and ChE levels were determined using a colorimetric method. We established that the ChE in the plasma of Griffons reflects mostly acetylcholinesterase as the dominant form. ChE levels in healthy Griffons are 0.601 ± 0.011 U/ml (mean ± SE), while Griffons with suspected or confirmed pesticide poisoning display much lower levels of ChE activity (typically <0.3 U/ml). We also characterized the age dependence of ChE activity, as well as differences among groups from different locations or origins. Our study provides a rapid diagnostic tool for the detection of exposure to organophosphate and carbamate pesticides that should facilitate the lifesaving treatment and the conservation of this species. Moreover, our protocols can be adapted to other species and geographical areas, addressing pesticide poisoning worldwide and contributing to the protection of endangered species and their ecological functions (e.g. sanitation by scavengers).

Preface: Cholinergic mechanisms: This is the Preface for the special issue "Cholinergic Mechanisms".

This special issue of the Journal of Neurochemistry, entitled "Cholinergic Mechanisms," presents 15 reviews and two original papers, which have been selected to cover the broad spectrum of topics and disciplines presented at the XVIth International Symposium on Cholinergic Mechanisms (ISCM-XVI), ranging from the molecular and the cellular to the clinical and the cognitive mechanisms of cholinergic transmission. The authors discuss recent developments in the field, for instance, the association of cholinergic transmission with a number of important neurological and neuromuscular diseases in the central and peripheral nervous systems.

Rhythmogenic networks are potently modulated by activation of muscarinic acetylcholine receptors in the rodent spinal cord.

Electrical stimulation of the spinal cord is a potent means for activating mammalian stepping in the absence of the descending control from the brain. Previously, we have shown that stimulation of pain delivering (Aδ) sacrocaudal afferents (SCA) has a powerful capacity to activate the sacral and lumbar rhythmogenic networks in the neonatal rodent spinal cord. Relatively little is known about the neural pathways involved in activation of the locomotor networks by Aδ afferents, on their mechanism of action and on the possibility to modulate their activity. We have shown that elevation of the endogenous level of acetylcholine at the sacral cord by blocking cholinesterase could modulate the SCA-induced locomotor rhythm in a muscarinic receptor-dependent mechanism. Here, we review these and more recent findings and report that controlled stimulation of SCA in the presence of muscarine is a potent activator of the locomotor network. The possible mechanisms involved in the muscarinic modulation of the locomotor rhythm are discussed in terms of the differential projections of sacral relay neurons, activated by SCA stimulation, to the lumbar locomotor rhythm generators, and to their target motoneurons. Altogether, our studies show that manipulations of cholinergic networks offer a simple and powerful means to control the activity of locomotor networks in the absence of supraspinal control. Cover Image for this issue: https://doi.org/10.1111/jnc.15079.

Fine Localization of Acetylcholinesterase in the Synaptic Cleft of the Vertebrate Neuromuscular Junction.

Acetylcholinesterase (AChE) is concentrated at cholinergic synapses, where it is a major factor in controlling the duration of transmitter action. The concentration and localization of AChE within the synaptic cleft are in keeping with the functional requirements of the particular type of synapse. The densities of synaptic AChE at various neuromuscular junctions (NMJs) had been evaluated by quantitative EM-autoradiography using radiolabeled probes. Yet, fundamental issues concerning the precise distribution and location of the enzyme in the cleft remained open: whether and to what extent synaptic AChE is associated with pre- or postsynaptic membranes, or with synaptic basal lamina (BL), and whether it occurs only in the primary cleft (PC) or also in postjunctional folds (PJFs). Nanogold-conjugates of fasciculin, an anticholinesterase polypeptide toxin, were prepared and used to label AChE at NMJs of mouse and frog muscles. Selective intense labeling was obtained at the NMJs, with gold-labeled AChE sites distributed over the BL in the PC and the PJFs. Quantitative analysis demonstrated that AChE sites are almost exclusively located on the BL rather than on pre- or postsynaptic membranes and are distributed in the PC and down the PJFs, with a defined pattern. This localization pattern of AChE is suggested to ensure full hydrolysis of acetylcholine (ACh) bouncing off receptors, thus eliminating its unnecessary detrimental reattachment.

Ascending pathways that mediate cholinergic modulation of lumbar motor activity.

Deciphering neuronal pathways that reactivate spinal central pattern generators (CPGs) and modulate the activity of spinal motoneurons in mammals in the absence of supraspinal control is important for understanding of neural control of movement and for developing novel therapeutic approaches to improve the mobility of spinal cord injury patients. Previously, we showed that the sacral and lumbar cholinergic system could potently modulate the locomotor CPGs in newborn rodents. Here, we review these and our more recent studies of sacral relay neurons with lumbar projections to the locomotor CPGs and to lumbar motoneurons and demonstrate that sacral and lumbar cholinergic components have the capacity to control the frequency of the locomotor CPGs and at the same time the motor output of the activated lumbar motoneurons during motor behavior. A model describing the suggested ascending sacro-lumbar connectivity involved in modulation of the locomotor rhythm by sacral cholinergic components is proposed and discussed. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Shaping the Output of Lumbar Flexor Motoneurons by Sacral Neuronal Networks.

The ability to improve motor function in spinal cord injury patients by reactivating spinal central pattern generators (CPGs) requires the elucidation of neurons and pathways involved in activation and modulation of spinal networks in accessible experimental models. Previously we reported on adrenoceptor-dependent sacral control of lumbar flexor motoneuron firing in newborn rats. The current work focuses on clarification of the circuitry and connectivity involved in this unique modulation and its potential use. Using surgical manipulations of the spinal gray and white matter, electrophysiological recordings, and confocal microscopy mapping, we found that methoxamine (METH) activation of sacral networks within the ventral aspect of S2 segments was sufficient to produce alternating rhythmic bursting (0.15-1 Hz) in lumbar flexor motoneurons. This lumbar rhythm depended on continuity of the ventral funiculus (VF) along the S2-L2 segments. Interrupting the VF abolished the rhythm and replaced it by slow unstable bursting. Calcium imaging of S1-S2 neurons, back-labeled via the VF, revealed that ∼40% responded to METH, mostly by rhythmic firing. All uncrossed projecting METH responders and ∼70% of crossed projecting METH responders fired with the concurrent ipsilateral motor output, while the rest (∼30%) fired with the contralateral motor output. We suggest that METH-activated sacral CPGs excite ventral clusters of sacral VF neurons to deliver the ascending drive required for direct rhythmic activation of lumbar flexor motoneurons. The capacity of noradrenergic-activated sacral CPGs to modulate the activity of lumbar networks via sacral VF neurons provides a novel way to recruit rostral lumbar motoneurons and modulate the output required to execute various motor behaviors. SIGNIFICANCE STATEMENT: Spinal central pattern generators (CPGs) produce the rhythmic output required for coordinating stepping and stabilizing the body axis during movements. Electrical stimulation and exogenous drugs can reactivate the spinal CPGs and improve the motor function in the absence of descending supraspinal control. Since the body-stabilizing sacral networks can activate and modulate the limb-moving lumbar circuitry, it is important to clarify the functional organization of sacral and lumbar networks and their linking pathways. Here we decipher the ascending circuitry linking adrenoceptor-activated sacral CPGs and lumbar flexor motoneurons, thereby providing novel insights into mechanisms by which sacral circuitry recruits lumbar flexors, and enhances the motor output during lumbar afferent-induced locomotor rhythms. Moreover, our findings might help to improve drug/electrical stimulation-based therapy to accelerate locomotor-based rehabilitation.

The sacral networks and neural pathways used to elicit lumbar motor rhythm in the rodent spinal cord.

Identification of neural networks and pathways involved in activation and modulation of spinal central pattern generators (CPGs) in the absence of the descending control from the brain is important for further understanding of neural control of movement and for developing innovative therapeutic approaches to improve the mobility of spinal cord injury patients. Activation of the hindlimb innervating segments by sacrocaudal (SC) afferent input and by specific application of neurochemicals to the sacral networks is feasible in the isolated spinal cord preparation of the newborn rat. Here we review our recent studies of sacral relay neurons with lumbar projections and evaluate their role in linking the sacral and thoracolumbar (TL) networks during different motor behaviors. Our major findings show that: (1) heterogeneous groups of dorsal, intermediate and ventral sacral-neurons with ventral and lateral ascending funicular projections mediate the activation of the locomotor CPGs through sacral sensory input; and (2) rhythmic excitation of lumbar flexor motoneurons, produced by bath application of alpha-1 adrenoceptor agonists to the sacral segments is mediated exclusively by ventral clusters of sacral-neurons with lumbar projections through the ventral funiculus.

The motor output of hindlimb innervating segments of the spinal cord is modulated by cholinergic activation of rostrally projecting sacral relay neurons.

Cholinergic networks have been shown to be involved in generation and modulation of the locomotor rhythmic pattern produced by the mammalian central pattern generators. Here, we show that changes in the endogenous levels of acetylcholine in the sacral segments of the isolated spinal cord of the neonatal rat modulate the locomotor-related output produced by stimulation of sacrocaudal afferents in muscarinic receptor-dependent mechanisms. Cholinergic components we found on sacral relay neurons with lumbar projections through the ventral and lateral funiculi are suggested to mediate this ascending cholinergic modulation. Our findings, possible mechanisms accounting for them, and their potential implications are discussed.

Neuroanatomical basis for cholinergic modulation of locomotor networks by sacral relay neurons with ascending lumbar projections.

Synaptic excitation by sacrocaudal afferent (SCA) input of sacral relay neurons projecting rostrally through the ventral white matter funiculi (VF neurons) is a potent activator of the hindlimb central pattern generators (CPGs) in rodent spinal cords lacking descending supraspinal control. Using electrophysiological recordings from the sacral and lumbar spinal segments, we show that the motor output of the lumbar segments produced by SCA stimulation is enhanced by exposing the sacral segments of the neonatal rat spinal cord to the acetylcholinesterase inhibitor edrophonium (EDR). Histochemical and immunostaining of the sacral cord reveals expression of acetylcholinesterase activity, ability to synthesize acetylcholine, and/or innervation by cholinergic synaptic inputs in significant proportions of fluorescently back-labeled sacral VF neurons. Moreover, the majority of the VF neurons express M2 muscarinic receptors, raising the possibility that the elevated acetylcholine levels resulting from inhibition of acetylcholinesterase act on such receptors. Indeed, sacral application of atropine or the M2 -type receptor antagonist methoctramine was found to reverse the effects of EDR. We suggest that variations in the sacral level of acetylcholine modulate the SCA-induced locomotor rhythm via muscarinic receptor-dependent mechanisms and that the modified activity of sacral VF neurons in the presence of an acetylcholinesterase inhibitor can be partially ascribed to the cholinergic components associated with them. Thus, pharmacological manipulations of the sacral cholinergic system may be used to modulate the locomotor-related motor output in the absence of descending supraspinal control.

Characterization of sacral interneurons that mediate activation of locomotor pattern generators by sacrocaudal afferent input.

Identification of the neural pathways involved in retraining the spinal central pattern generators (CPGs) by afferent input in the absence of descending supraspinal control is feasible in isolated rodent spinal cords where the locomotor CPGs are potently activated by sacrocaudal afferent (SCA) input. Here we study the involvement of sacral neurons projecting rostrally through the ventral funiculi (VF) in activation of the CPGs by sensory stimulation. Fluorescent labeling and immunostaining showed that VF neurons are innervated by primary afferents immunoreactive for vesicular glutamate transporters 1 and 2 and by intraspinal neurons. Calcium imaging revealed that 55% of the VF neurons were activated by SCA stimulation. The activity of VF neurons and the sacral and lumbar CPGs was abolished when non-NMDA receptors in the sacral segments were blocked by the antagonist CNQX. When sacral NMDA receptors were blocked by APV, the sacral CPGs were suppressed, VF neurons with nonrhythmic activity were recruited and a moderate-drive locomotor rhythm developed during SCA stimulation. In contrast, when the sacral CPGs were activated by SCA stimulation, rhythmic and nonrhythmic VF neurons were recruited and the locomotor rhythm was most powerful. The activity of 73 and 27% of the rhythmic VF neurons was in-phase with the ipsilateral and contralateral motor output, respectively. Collectively, our studies indicate that sacral VF neurons serve as a major link between SCA and the hindlimb CPGs and that the ability of SCA to induce stepping can be enhanced by the sacral CPGs. The nature of the ascending drive to lumbar CPGs, the identity of subpopulations of VF neurons, and their potential role in activating the locomotor rhythm are discussed.

Changes in acetylcholinesterase in experimental autoimmune myasthenia gravis and in response to treatment with a specific antisense.

Controlled regulation of synaptic nicotinic acetylcholine receptors (AChRs) and acetylcholinesterase (AChE), together with maintenance of a dynamic balance between them, is a requirement for proper function of cholinergic synapses. In the present study we assessed whether pathological changes in AChR perturb this balance, and whether such changes can be corrected. We studied the influence of AChR loss, caused by experimental autoimmune myasthenia gravis (EAMG), on muscle AChE, as well as the reciprocal effect of an antisense targeted towards AChE on both AChR and AChE at the neuromuscular synapse. The extensor digitorum longus (EDL) muscles of EAMG Lewis rats were isolated, and AChE levels and isoform compositions were examined. Although AChE levels in the muscles of healthy and EAMG rats were similar, marked changes were observed in isoform composition. Healthy EDL muscles contained globular (G(1,2) , G(4) ) and asymmetric (primarily A(12) ) isoforms. G(1,2) -AChE was significantly reduced in EAMG muscles, whereas both G(4) - and A(12) -AChE remained unchanged. Treatment of EAMG rats with the antisense EN101 resulted in decreased total muscle AChE, with recovery in G(1,2) and reduction in A(12) -AChE. AChE/AChR ratios were determined at the neuromuscular junctions (NMJ). The decrease in AChR levels that occurred as the disease progressed resulted in a dramatic increase in this ratio, and a significant recovery towards normal ratios occurred after EN101 treatment. This improvement was primarily due to increased synaptic AChR content. Our findings emphasise the tight connection between AChR and AChE at the myasthenic NMJ, and the importance of the AChE/AChR ratio in maintaining the required cholinergic balance.

Serum cholinesterases are differentially regulated in normal and dystrophin-deficient mutant mice.

The cholinesterases, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) (pseudocholinesterase), are abundant in the nervous system and in other tissues. The role of AChE in terminating transmitter action in the peripheral and central nervous system is well understood. However, both knowledge of the function(s) of the cholinesterases in serum, and of their metabolic and endocrine regulation under normal and pathological conditions, is limited. This study investigates AChE and BChE in sera of dystrophin-deficient mdx mutant mice, an animal model for the human Duchenne muscular dystrophy (DMD) and in control healthy mice. The data show systematic and differential variations in the concentrations of both enzymes in the sera, and specific changes dictated by alteration of hormonal balance in both healthy and dystrophic mice. While AChE in mdx-sera is elevated, BChE is markedly diminished, resulting in an overall cholinesterase decrease compared to sera of healthy controls. The androgen testosterone (T) is a negative modulator of BChE, but not of AChE, in male mouse sera. T-removal elevated both BChE activity and the BChE/AChE ratio in mdx male sera to values resembling those in healthy control male mice. Mechanisms of regulation of the circulating cholinesterases and their impairment in the dystrophic mice are suggested, and clinical implications for diagnosis and treatment are considered.