Alterations in the motor neuron–renshaw cell circuit in the Sod1G93A mouse model

Motor neurons become hyperexcitable during progression of amyotrophic lateral sclerosis (ALS). This abnormal firing behavior has been explained by changes in their membrane properties, but more recently it has been suggested that changes in premotor circuits may also contribute to this abnormal activity. The specific circuits that may be altered during development of ALS have not been investigated. Here we examined the Renshaw cell recurrent circuit that exerts inhibitory feedback control on motor neuron firing. Using two markers for Renshaw cells (calbindin and cholinergic nicotinic receptor subunit alpha2 [Chrna2]), two general markers for motor neurons (NeuN and vesicular acethylcholine transporter [VAChT]), and two markers for fast motor neurons (Chondrolectin and calcitonin‐related polypeptide alpha [Calca]), we analyzed the survival and connectivity of these cells during disease progression in the Sod1^G93A mouse model. Most calbindin‐immunoreactive (IR) Renshaw cells survive to end stage but downregulate postsynaptic Chrna2 in presymptomatic animals. In motor neurons, some markers are downregulated early (NeuN, VAChT, Chondrolectin) and others at end stage (Calca). Early downregulation of presynaptic VAChT and Chrna2 was correlated with disconnection from Renshaw cells as well as major structural abnormalities of motor axon synapses inside the spinal cord. Renshaw cell synapses on motor neurons underwent more complex changes, including transitional sprouting preferentially over remaining NeuN‐IR motor neurons. We conclude that the loss of presynaptic motor axon input on Renshaw cells occurs at early stages of ALS and disconnects the recurrent inhibitory circuit, presumably resulting in diminished control of motor neuron firing. J. Comp. Neurol. 521:1449–1469, 2013. © 2012 Wiley Periodicals, Inc.     

Distribution of the vesicular acetylcholine transporter (VAChT) in the central and peripheral nervous systems of the rat

Expression of the acetylcholine biosynthetic enzyme choline acetyltransferase (ChAT), the vesicular acetylcholine transporter (VAChT), and the high-affinity plasma membrane choline transporter uniquely defines the cholinergic phenotype in the mammalian central (CNS) and peripheral (PNS) nervous systems. The distribution of cells expressing the messenger RNA encoding the recently cloned VAChT in the rat CNS and PNS is described here. The pattern of expression of VAChT mRNA is consistent with anatomical, pharmacological, and histochemical information on the distribution of functional cholinergic neurons in the brain and peripheral tissues of the rat. VAChT mRNA-containing cells are present in brain areas, including neocortex and hypothalamus, in which the existence of cholinergic neurons has been the subject of debate. The demonstration that VAChT is a completely adequate marker for cholinergic neurons should allow the systematic delineation of cholinergic synapses in the rat nervous system when antibodies directed to this protein are available.     

Peripheral type of choline acetyltransferase: biological and evolutionary implications for novel mechanisms in cholinergic system

The peripheral type of choline acetyltransferase (pChAT) is an isoform of the well-studied common type of choline acetyltransferase (cChAT), the synthesizing enzyme of acetylcholine. Since pChAT arises by exons skipping, its amino acid sequence is similar to that of cChAT, except the lack of a continuous peptide sequence encoded by all the four exons from 6 to 9. While cChAT expression has been observed in both the central and peripheral nervous systems, pChAT is preferentially expressed in the peripheral nervous system.pChAT appears to be a reliable marker for the visualization of peripheral cholinergic neurons and their processes, whereas other conventional markers including cChAT have not been used successfully for it. In mammals like rodents, pChAT immunoreactivity has been observed in most, if not all, physiologically identified peripheral cholinergic structures such as all parasympathetic postganglionic neurons and most neurons of the enteric nervous system. In addition, pChAT has been found in many peripheral neurons that are derived from the neural crest. These include sensory neurons of the trigeminal ganglion and the dorsal root ganglion, and sympathetic postganglionic neurons. Recent studies moreover indicate that pChAT, as well as cChAT, appears ubiquitously expressed among various species not only of vertebrate mammals but also of invertebrate mollusks. This finding implies that the alternative splicing mechanism to generate pChAT and cChAT has been preserved during evolution, probably for some functional benefits.     

Reduced density of neuropeptide Y neurons in the somatosensory cortex of old male and female rats: relation to cholinergic depletion and recovery after nerve growth factor treatment

Synthesis of neuropeptide Y in the neocortex and activity of the basalocortical cholinergic system are both reduced in the aging brain. We hypothesized that, by stimulating the activity of the basal forebrain cholinergic neurons, nerve growth factor might also be capable of restoring the synthesis of neuropeptide Y in cortical neurons. Old male and female rats were intraventricularly infused with nerve growth factor for 14 days and their brains were analyzed in order to quantify the densities of neuropeptide Y-immunoreactive neurons and of fiber varicosities stained for vesicular acetylcholine transporter protein in layers II/III, V and VI of the primary somatosensory barrel-field cortex. The areal densities of neuropeptide Y neurons and of vesicular acetylcholine transporter protein varicosities in all cortical laminae were found to be dramatically decreased in old rats when compared with young rats. However, infusions of nerve growth factor, known to exert a powerful trophic effect upon cortically projecting cholinergic neurons, have led to considerable recovery of vesicular acetylcholine transporter protein-positive terminal fields, which was paralleled by complete restoration of function in neuropeptide Y-producing neurons. With respect to the gender differences, although the density of cortical neuropeptide Y neurons was found to be significantly higher in young females than in young males and the opposite was true for vesicular acetylcholine transporter protein-positive varicosities, the general pattern of age- and treatment-related changes in these neurochemical markers was similar in both sexes. Overall, the age- and treatment-related variations in the density of cortical neuropeptide Y cells were found to correlate with those observed in the density of vesicular acetylcholine transporter protein varicosities. These results lend support to the idea that there is a causal relationship between age-related changes in cortical cholinergic and neuropeptide Y-ergic neurotransmitter systems.     

Neurotransmitter segregation: functional and plastic implications

Synaptic cotransmission is the ability of neurons to use more than one transmitter to convey synaptic signals. Cotransmission was originally described as the presence of a classic transmitter, which conveys main signal, along one or more cotransmitters that modulate transmission, later on, it was found cotransmission of classic transmitters. It has been generally accepted that neurons store and release the same set of transmitters in all their synaptic processes. However, some findings that show axon endings of individual neurons storing and releasing different sets of transmitters, are not in accordance with this assumption, and give support to the hypothesis that neurons can segregate transmitters to different synapses. Here, we review the studies showing segregation of transmitters in invertebrate and mammalian central nervous system neurons, and correlate them with our results obtained in sympathetic neurons. Our data show that these neurons segregate even classic transmitters to separated axons. Based on our data we suggest that segregation is a plastic phenomenon and responds to functional synaptic requirements, and to 'environmental' cues such as neurotrophins. We propose that neurons have the machinery to guide the different molecules required in synaptic transmission through axons and sort them to different axon endings. We believe that transmitter segregation improves neuron interactions during cotransmission and gives them selective and better control of synaptic plasticity.     

The cholinergic gene locus in amphioxus: molecular characterization and developmental expression patterns.

The cholinergic gene locus (CGL), consisting of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT) gene, encodes two specific cholinergic neuronal markers used extensively to study cholinergic transmission. In the present work, we isolated the amphioxus homologs of VAChT and ChAT and examined their expression during development. Analysis of the 5' untranslated region of VAChT and ChAT suggests that the splicing of the VAChT/ChAT mRNA has been evolutionarily conserved in amphioxus and mammals. By double whole-mount in situ hybridization, we demonstrate that VAChT and ChAT are coexpressed in the same cells. They are first expressed in four pairs of differentiating cells in the neural plate. Their later expression is primarily in the anterior nerve cord in several types of motoneurons, some of the interneurons and in the receptor cells of the larval ocellus.     

The basal forebrain cholinergic system in aging and dementia. Rescuing cholinergic neurons from neurotoxic amyloid-β42 with memantine

The dysfunction and loss of basal forebrain cholinergic neurons and their cortical projections are among the earliest pathological events in the pathogenesis of Alzheimer's disease (AD). The evidence pointing to cholinergic impairments come from studies that report a decline in the activity of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE), acetylcholine (ACh) release and the levels of nicotinic and muscarinic receptors, and loss of cholinergic basal forebrain neurons in the AD brain. Alzheimer's disease pathology is characterized by an extensive loss of synapses and neuritic branchings which are the dominant scenario as compared to the loss of the neuronal cell bodies themselves. The appearance of cholinergic neuritic dystrophy, i.e. aberrant fibers and fiber swelling are more and more pronounced during brain aging and widely common in AD. When taking amyloid-β (Aβ) deposition as the ultimate causal factor of Alzheimer's disease the role of Aβ in cholinergic dysfunction should be considered. In that respect it has been stated that ACh release and synthesis are depressed, axonal transport is inhibited, and that ACh degradation is affected in the presence of Aβ peptides. β-Amyloid peptide 1-42, the principal constituent of the neuritic plaques seen in AD patients, is known to trigger excess amount of glutamate in the synaptic cleft by inhibiting the astroglial glutamate transporter and to increase the intracellular Ca²⁺ level. Based on the glutamatergic overexcitation theory of AD progression, the function of NMDA receptors and treatment with NMDA antagonists underlie some recent therapeutic applications. Memantine, a moderate affinity uncompetitive NMDA receptor antagonist interacts with its target only during states of pathological activation but does not interfere with the physiological receptor functions. In this study the neuroprotective effect of memantine on the forebrain cholinergic neurons against Aβ42 oligomers-induced toxicity was studied in an in vivo rat dementia model. We found that memantine rescued the neocortical cholinergic fibers originating from the basal forebrain cholinergic neurons, attenuated microglial activation around the intracerebral lesion sides, and improved attention and memory of Aβ42-injected rats exhibiting impaired learning and loss of cholinergic innervation of neocortex.     

Role of endogenous nicotinic signaling in guiding neuronal development

Spontaneous nicotinic cholinergic activity is widespread in the developing nervous system. One of the major components mediating this activity is the nicotinic acetylcholine receptor with alpha7 subunits (alpha7-nAChR) and high relative calcium permeability. We recently reported that alpha7-nAChRs co-localize in part with GABA(A) receptors during development, and the sites become co-innervated by cholinergic and GABAergic terminals. Patch-clamp recording either from embryonic chick ciliary ganglion neurons or from early postnatal mouse hippocampal interneurons reveals that alpha7-nAChR activation can impose a rapid and reversible decrease in GABA(A) receptor responses. The effect extends to GABAergic synaptic currents, and depends on intracellular calcium, calcium/calmodulin-dependent protein kinase II, and MAP kinase in the postsynaptic cell. Over the longer term, nicotinic activity has a more profound effect: it determines the time during development when GABAergic signaling converts from excitation to inhibition. It does this by changing the pattern of chloride transporters to establish the mature chloride gradient required for inhibitory GABAergic responses. The excitatory phase of GABAergic signaling is critical for proper development and integration of neurons into circuits. By driving the conversion of GABAergic signaling, nicotinic activity not only terminates one set of developmental instructions, but also initiates another by collaborating with GABAergic inhibition to impose new instructions. The results reveal a multi-layered pattern of activity-dependent controls in development and indicate the significance of nicotinic signaling in shaping these events.     

Innervation of orexin/hypocretin neurons by GABAergic, glutamatergic or cholinergic basal forebrain terminals evidenced by immunostaining for presynaptic vesicular transporter and postsynaptic scaffolding proteins

Orexin/hypocretin (Orx) neurons are critical for the maintenance of waking in association with behavioral arousal and postural muscle tone, since with their loss narcolepsy with cataplexy occurs. Given that basal forebrain (BF) neurons project to the hypothalamus and play important diverse roles in sleep/wake states, we sought to determine whether acetylcholine (ACh), glutamate (Glu), and/or GABA-releasing BF neurons innervate and could thereby differentially regulate the Orx neurons. From discrete injections of biotinylated dextran amine (BDA, 10,000 MW) into the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) in the rat, BDA-labeled fibers projected to the lateral hypothalamus (LH), perifornical area (PF), and dorsomedial hypothalamus (DMH), where approximately 41%, approximately 11%, and 9% of Orx-positive (+) neurons were respectively contacted in each region. Employing triple fluorescent staining for Orx, BDA, and presynaptic vesicular (V) transporters (T), we found that only 4% of the innervated Orx+ neurons in the LH were contacted by BDA+[VAChT+] terminals, whereas approximately 31% and approximately 67% were respectively contacted by BDA+[VGluT2+] and BDA+[VGAT+] terminals. In 3D-rendered and rotated confocal images, we confirmed the latter contacts and examined staining for postsynaptic proteins PSD-95, a marker for glutamatergic synapses, and gephyrin, a marker for GABAergic synapses, that were located on Orx+ neurons facing BDA-labeled terminals in approximately 20% and approximately 50% of contacts, respectively. With such synaptic input, BF glutamatergic neurons can excite Orx neurons and thus act to maintain behavioral arousal with muscle tone, whereas GABAergic neurons can inhibit Orx neurons and thus promote behavioral quiescence and sleep along with muscle atonia.