Increased Acetylcholine Synthesis and Release in Brains of Cats with GM1 Gangliosidosis

Cholinergic processes were measured in motor cortex, hippocampus, and striatum of cats in the terminal stages of GM1 gangliosidosis and compared to those of control cats. The greatest difference observed was elevation in the rate of K+-stimulated release of acetylcholine (ACh) from brain slices prepared from affected cats. The K+-stimulated release of endogenous ACh was increased by 31-43% and of newly synthesized ACh by 19-80% in brain slices from different brain regions. All regions that were examined were affected but the greatest effects occurred in cortex. The rate of synthesis of ACh was elevated in cortical and hippocampal slices. Choline acetyltransferase activity in brain regions of cats with GM1 gangliosidosis was not significantly different from that in controls, whereas high-affinity choline transport in cortical synaptosomes was elevated. Muscarinic receptor binding sites were reduced in the cortex, hippocampus, and striatum of GM1 mutant cats, whereas the apparent affinity was not altered. These results indicate that there are major alterations of cholinergic function in the brains of cats with GM1 gangliosidosis.     

Lesions of the Basal forebrain cholinergic system impair task acquisition and abolish cortical plasticity associated with motor skill learning

The contribution of the basal forebrain cholinergic system in mediating plasticity of cortical sensorimotor representations was examined in the context of normal learning. The effects of specific basal forebrain cholinergic lesions upon cortical reorganization associated with learning a skilled motor task were investigated, addressing, for the first time, the functional consequences of blocking cortical map plasticity. Results demonstrate that disrupting basal forebrain cholinergic function disrupts cortical map reorganization and impairs motor learning. Cholinergic lesions do not impair associative fear learning or overall sensorimotor function. These results support the hypothesis that the basal forebrain cholinergic system may be specifically implicated in forms of learning requiring plasticity of cortical representations.     

Changes in cortical acetyl-CoA metabolism after selective basal forebrain cholinergic degeneration by 192IgG-saporin

The aim of the present study was to reveal whether reduced cortical cholinergic input affects the acetyl-CoA metabolism in cholinoceptive cortical target regions which may play a causative role for the deficits in cerebral glucose metabolism observed in Alzheimer's disease. The effect of cortical cholinergic denervation produced by a single intracerebroventricular application of the cholinergic immunotoxin 192IgG-saporin, on activities of pyruvate dehydrogenase and adenosine triphosphate (ATP)-citrate lyase as well as on the level of synaptoplasmic and mitochondrial acetyl-CoA and acetylcholine release in cortical target regions was studied. Cholinergic lesion produced 83%, 72% and 32% decreases in the activities of choline acetyltransferase, acetylcholinesterase and ATP-citrate lyase in nerve terminals isolated from rat brain cortex, respectively, but no change in pyruvate dehydrogenase activity. Spontaneous and Ca2+-evoked acetylcholine release from synaptosomes was inhibited by 76% and 73%, respectively, following immunolesion. The lesion-induced 39% decrease of acetyl-CoA level in synaptosomal mitochondria was accompanied by 74% increase in synaptoplasmic fraction. Levels of acetyl-CoA and CoASH assayed in fraction of whole brain mitochondria from lesioned cortex were 61% and 48%, respectively, higher as compared to controls. The data suggest a preferential localization of ATP-citrate lyase in cholinergic nerve terminals, where it may contribute to the transport of acetyl-CoA from the mitochondrial to the cytoplasmic compartment. They provide evidence on differential distribution of acetyl-CoA in subcellular compartments of cholinergic and non-cholinergic nerve terminals. There are also indications that cholinergic activity affects acetyl-CoA level and its intracellular distribution in glial and other non-cholinergic cortical cells.     

Basal forebrain cholinergic input is not essential for lesion-induced plasticity in mature auditory cortex

The putative role of the basal forebrain cholinergic system in mediating lesion-induced plasticity in topographic cortical representations was investigated. Cholinergic immunolesions were combined with unilateral restricted cochlear lesions in adult cats, demonstrating the consequence of cholinergic depletion on lesion-induced plasticity in primary auditory cortex (AI). Immunolesions almost eliminated the cholinergic input to AI, while cochlear lesions produced broad high-frequency hearing losses. The results demonstrate that the near elimination of cholinergic input does not disrupt reorganization of the tonotopic representation of the lesioned (contralateral) cochlea in AI and does not affect the normal representation of the unlesioned (ipsilateral) cochlea. It is concluded that cholinergic basal forebrain input to AI is not essential for the occurrence of lesion-induced plasticity in AI.     

Time-Related Cortical Amino Acid Changes After Basal Forebrain Lesion: A Microdialysis Study

Abstract: Cholinergic basal forebrain (BF) lesions in experimental animals have been used as a potential model for cholinergic deficits in cortex and hippocampus that occur in normal aging and Alzheimer's disease (AD). Glutamatergic cortical neurons are also affected in AD and could be part of the neurodegenerative process. In the present study, the effect of bilateral BF lesion with ibotenic acid microinjection on cortical extracellular amino acid levels was determined. Samples were collected every 20 min with microdialysis probes in awake, freely moving rats under basal and potassium stimulation conditions and measured by HPLC with fluorescence detection. Microdialysis experiments were performed 13 days, 21 days, and 30 days after BF lesion. The effectiveness of the lesion was shown by a significant 30% depletion in acetyl-CoA:choline O -acetyltransferase (EC 2.3.1.6) activity in the frontal cortex. Under basal conditions at 13 days only extracellular levels of taurine (Tau) and Glu were significantly reduced. Tau and Glu levels were recovered after 21 days and 30 days, respectively. In contrast, increase in Gly levels reaches its significance only at 30 days after lesion. Significant increases of Gln levels were observed at 21 days and 30 days. Asp and Ser levels remained constant throughout the period studied. Potassium stimulation led to increased Asp, Glu, Gly, and Tau levels, whereas Gln content decreased and Ser remained unaltered. As Ser is not believed to be a neurotransmitter, its lack of variation in any of the experimental conditions studied supports specific neuronal changes of the other amino acids. Results are discussed with reference to data observed in AD patients and possible mechanisms underlying the changes are suggested.     

Increases in cholinergic neurotransmission measured by using choline-sensitive microelectrodes: enhanced detection by hydrolysis of acetylcholine on recording sites?

Previous experiments demonstrated that second-based transient increases in choline concentrations measured by electrodes coated with choline oxidase (ChOx) and the amperometric detection of hydrogen peroxide validly indicate the depolarization-dependent release of acetylcholine (ACh) and its hydrolysis by endogenous acetylcholinesterase (AChE). Therefore, choline-sensitive microelectrodes have become valuable tools in neuropharmacological and behavioral research. The present experiments were designed to test the possibility that co-immobilization of ChOx plus AChE on recording sites increases the level of detection for evoked ACh release in the brain. If newly released ACh is not completely hydrolyzed by endogenous AChE and capable of reaching the extracellular space, currents recorded via sites equipped with both enzymes should be greater when compared with sites coated with ChOx only. Pairs of platinum-recording sites were coated either with AChE plus ChOx or ChOx alone. Potassium or nicotine-evoked currents were recorded throughout the entire dorsal–ventral extent of the medial prefrontal cortex (mPFC). The amplitudes of evoked cholinergic signals did not differ significantly between AChE + ChOx and ChOx-only coated recording sites. Additional experiments controlling for several potential confounds suggested that, in vivo, ACh levels ≥150 fmol were detected by recordings sites featuring dual enzyme coating. Collectively, these results indicate that co-coating of microelectrodes with AChE does not enhance the detection of cholinergic activity in the cortex compared with measurements via recording sites coated only with ChOx.     

Muscarinic and Nicotinic Modulation of Thalamo-Prefrontal Cortex Synaptic Pasticity In Vivo

The mediodorsal nucleus of the thalamus (MD) is a rich source of afferents to the medial prefrontal cortex (mPFC). Dysfunctions in the thalamo-prefrontal connections can impair networks implicated in working memory, some of which are affected in Alzheimer disease and schizophrenia. Considering the importance of the cholinergic system to cortical functioning, our study aimed to investigate the effects of global cholinergic activation of the brain on MD-mPFC synaptic plasticity by measuring the dynamics of long-term potentiation (LTP) and depression (LTD) in vivo. Therefore, rats received intraventricular injections either of the muscarinic agonist pilocarpine (PILO; 40 nmol/µL), the nicotinic agonist nicotine (NIC; 320 nmol/µL), or vehicle. The injections were administered prior to either thalamic high-frequency (HFS) or low-frequency stimulation (LFS). Test pulses were applied to MD for 30 min during baseline and 240 min after HFS or LFS, while field postsynaptic potentials were recorded in the mPFC. The transient oscillatory effects of PILO and NIC were monitored through recording of thalamic and cortical local field potentials. Our results show that HFS did not affect mPFC responses in vehicle-injected rats, but induced a delayed-onset LTP with distinct effects when applied following PILO or NIC. Conversely, LFS induced a stable LTD in control subjects, but was unable to induce LTD when applied after PILO or NIC. Taken together, our findings show distinct modulatory effects of each cholinergic brain activation on MD-mPFC plasticity following HFS and LFS. The LTP-inducing action and long-lasting suppression of cortical LTD induced by PILO and NIC might implicate differential modulation of thalamo-prefrontal functions under low and high input drive.     

Axonal varicosity density as an index of local neuronal interactions

Diffuse transmission is an important non-synaptic communication mode in the cerebral neocortex, in which neurotransmitters released from en passant varicosities interact with surrounding cells. In a previous study we have shown that the cholinergic axonal segments which were in the microproximity with dopaminergic fibers possessed a greater density of en passant varicosities compared to more distant segments, suggesting an activity-dependent level of en passant varicosities in the axonal zone of interaction. To further evaluate this plastic relationship, the density of cholinergic varicosities was quantified on fiber segments within the microproximity of activated or non-activated pyramidal cells of the prefrontal cortex (mPFC). Repetitive 14 days patterned visual stimulation paired with an electrical stimulation of the cholinergic fibers projecting to the mPFC from the HDB was performed to induce persistent axonal plastic changes. The c-Fos early gene immunoreactivity was used as a neuronal activity marker of layer V pyramidal cells, labelled with anti-glutamate transporter EAAC1. Cholinergic fibers were labeled with anti-ChAT (choline acetyltransferase) immunostaining. The density of ChAT+ varicosities on and the length of fiber segments within the 3 μm microproximity of c-Fos positive/negative pyramidal cells were evaluated on confocal images. More than 50% of the pyramidal cells in the mPFC were c-Fos immunoreactive. Density of ChAT+ varicosities was significantly increased within 3 μm vicinity of activated pyramidal cells (0.50±0.01 per μm of ChAT+ fiber length) compared to non-activated cells in this group (0.34±0.001; p≤0.05) or control rats (0.32±0.02; p≤0.05). Different types of stimulation (visual, HDB or visual/HDB) induced similar increase of the density of ChAT+ varicosities within microproximity of activated pyramidal cells. This study demonstrated at the subcellular level an activity-dependent enrichment of ChAT+ varicosities in the axonal zone of interaction with other neuronal elements.     

Single units in the medial prefrontal cortex with anxiety-related firing patterns are preferentially influenced by ventral hippocampal activity

The medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC) functionally interact during innate anxiety tasks. To explore the consequences of this interaction, we examined task-related firing of single units from the mPFC of mice exploring standard and modified versions of the elevated plus maze (EPM), an innate anxiety paradigm. Hippocampal local field potentials (LFPs) were simultaneously monitored. The population of mPFC units distinguished between safe and aversive locations within the maze, regardless of the nature of the anxiogenic stimulus. Strikingly, mPFC units with stronger task-related activity were more strongly coupled to theta-frequency activity in the vHPC LFP. Lastly, task-related activity was inversely correlated with behavioral measures of anxiety. These results clarify the role of the vHPC-mPFC circuit in innate anxiety and underscore how specific inputs may be involved in the generation of behaviorally relevant neural activity within the mPFC.     

Cholinergic regulation of the activity of somatosensory cortex neurons during early postnatal ontogeny of the cat

In 11-14 days kittens, about 20% of neurones in the somatosensory cortical zone react to stimulation of subpallidal region which is a source of cholinergic projections to the cerebral cortex. The effect of subpallidal region stimulation is reproduced in case of microiontophoretic acetylcholine application and blocked by atropine what points to its cholinergic nature. Cholinergic stimulation causes inhibition of the background and evoked activities of the cortical neurones while, as it is known, in adult cats, acetylcholine mainly stimulates a reaction of activation. It is postulated that in kittens at the end of the second week of postnatal development, cholinergic innervation of the cortex significantly differs from the definitive one by its quantitative and functional parameters.     

A possible mechanism of participation of dopaminergic cells and striatal cholinergic interneurons in the conditioned selection of motor activity

A possible mechanism of participation of cholinergic striatal interneurons and dopaminergic cells in conditioned selection of a certain types of motor activity is proposed. This selection is triggered by simultaneous increase in the activity of dopaminergic cells and a pause in the activity of cholinergic interneurons in response to a conditioned stimulus. This pause is promoted by activation of striatal inhibitory interneurons and action of dopamine at D2 receptors on cholinergic cells. Opposite changes in dopamine and acetylcholine concentration synergistically modulate the efficacy of corticostriatal inputs, modulation rules for the "strong" and "weak" corticostriatal inputs are opposite. Subsequent reorganization of neuronal firing in the loop cortex--basal ganglia--thalamus--cortex results in amplification of activity of the group of cortical neurons that strongly activate striatal cells, and simultaneous suppression of activity of another group of cortical neurons that weakly activate striatal cells. These changes can underlie a conditioned selection of motor activity performed with involvement of the motor cortex. As follows from the proposed model, if the time delay between conditioned and unconditioned stimuli does not exceed the latency of responses of dopaminergic and cholinergic cells (about 100 ms), conditioned selection of motor activity and learning is problematic.     

Paying attention with the latest technology

In this issue of Neuron, Parikh et al. utilize biosensors to probe the cholinergic system in freely moving rats performing cue-detection/reward delivery tasks. They show that cue-evoked cholinergic activity in the medial prefrontal cortex is associated with cue detection and not reward delivery. We discuss the implications of their research in behavioral neuroscience.     

Some cholinergic themes related to Alzheimer's disease: Synaptology of the nucleus basalis,location of m2 receptors,interactions with amyloid metabolism,and perturbations of cortical plasticity

Cholinergic neurons in the nucleus basalis of Meynert (nbM) receive cholinergic, GABAergic and monoaminergic synapses. Only few of these neurons display the sort of intense m2 immunoreactivity that would be expected if they were expressing m2 as their presynaptic autoreceptor. The depletion of cortical m2 in Alzheimer's disease (AD) appears to reflect the loss of presynaptic autoreceptors located on incoming axons from the nucleus basalis of Meynert (nbM) and also the loss of postsynaptic receptors located on a novel group of nitric oxide producing interstitial neurons in the cerebral cortex. The defect of cholinergic transmission in AD may enhance the neurotoxicity of amyloid β, leading to a vicious cycle which can potentially accelerate the pathological process. Because acetylcholine plays a critical role in regulating axonal growth and synaptic remodeling, the cholinergic loss in AD can perturb cortical plasticity so as to undermine the already fragile compensatory reserve of the aging cerebral cortex.     

Changes in choline acetyltransferase activity and high-affinity choline uptake, but not in acetylcholinesterase activity and muscarinic cholinergic receptors, in rat somatosensory cortex after sciatic nerve injury

Selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, high-affinity choline uptake) were studied in the hindlimb representation areas of the rat somatosensory cortex and within the visual cortex 1 to 63 days after unilateral transection of the sciatic nerve. In the contralateral somatosensory cortex, peripheral deafferentation resulted in a significant reduction of choline acetyltransferase activity (by 15%) 3 days after sciatic nerve injury, and in a significant reduction of high-affinity choline uptake (by 30%) 1 day after nerve transection, in comparison to untreated control rats. Investigations in individual cortical layers revealed that the decrease of both choline acetyltransferase activity and high-affinity choline uptake sites was mainly due to reductions in cortical layer V. Acetylcholinesterase activity and [3H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors were not affected by unilateral transection of the sciatic nerve. In the ipsilateral somatosensory cortex, as well as in the visual cortex at both cortical hemispheres, no significant changes in the cholinergic parameters studied could be detected. The data indicate that peripheral deafferentation of the somatosensory cortex results in a transient change of presynaptic cholinergic parameters within the affected somatosensory area as early as 1 to 3 days after the lesion; thus, they emphasize the involvement of cholinergic mechanisms in cortical reorganizational events.     

Changes in Choline Acetyltransferase Activity and High-Affinity Choline Uptake,but Not in Acetylcholinesterase Activity and Muscarinic Cholinergic Receptors,in Rat Somatosensory Cortex after Sciatic Nerve Injury

Selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, high-affinity choline uptake) were studied in the hindlimb representation areas of the rat somatosensory cortex and within the visual cortex 1 to 63 days after unilateral transection of the sciatic nerve. In the contralateral somatosensory cortex, peripheral deafferentation resulted in a significant reduction of choline acetyltransferase activity (by 15%) 3 days after sciatic nerve injury, and in a significant reduction of high-affinity choline uptake (by 30%) 1 day after nerve transection, in comparison to untreated control rats. Investigations in individual cortical layers revealed that the decrease of both choline acetyltransferase activity and high-affinity choline uptake sites was mainly due to reductions in cortical layer V. Acetylcholinesterase activity and [³H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors were not affected by unilateral transection of the sciatic nerve. In the ipsilateral somatosensory cortex, as well as in the visual cortex at both cortical hemispheres, no significant changes in the cholinergic parameters studied could be detected. The data indicate that peripheral deafferentation of the somatosensory cortex results in a transient change of presynaptic cholinergic parameters within the affected somatosensory area as early as 1 to 3 days after the lesion; thus, they emphasize the involvement of cholinergic mechanisms in cortical reorganizational events.     

Long-term Relationships between Cholinergic Tone, Synchronous Bursting and Synaptic Remodeling

Cholinergic neuromodulation plays key roles in the regulation of neuronal excitability, network activity, arousal, and behavior. On longer time scales, cholinergic systems play essential roles in cortical development, maturation, and plasticity. Presumably, these processes are associated with substantial synaptic remodeling, yet to date, long-term relationships between cholinergic tone and synaptic remodeling remain largely unknown. Here we used automated microscopy combined with multielectrode array recordings to study long-term relationships between cholinergic tone, excitatory synapse remodeling, and network activity characteristics in networks of cortical neurons grown on multielectrode array substrates. Experimental elevations of cholinergic tone led to the abrupt suppression of episodic synchronous bursting activity (but not of general activity), followed by a gradual growth of excitatory synapses over hours. Subsequent blockage of cholinergic receptors led to an immediate restoration of synchronous bursting and the gradual reversal of synaptic growth. Neither synaptic growth nor downsizing was governed by multiplicative scaling rules. Instead, these occurred in a subset of synapses, irrespective of initial synaptic size. Synaptic growth seemed to depend on intrinsic network activity, but not on the degree to which bursting was suppressed. Intriguingly, sustained elevations of cholinergic tone were associated with a gradual recovery of synchronous bursting but not with a reversal of synaptic growth. These findings show that cholinergic tone can strongly affect synaptic remodeling and synchronous bursting activity, but do not support a strict coupling between the two. Finally, the reemergence of synchronous bursting in the presence of elevated cholinergic tone indicates that the capacity of cholinergic neuromodulation to indefinitely suppress synchronous bursting might be inherently limited.     

High-Affinity Choline Transport Regulation by Drug Administration During Postnatal Development

High-affinity choline transport sites specifically bind [3H]hemicholinium-3. Hemicholinium-3 binding sites are regulated by in vivo drug treatments in the same manner as these drugs alter acetylcholine release and high-affinity choline transport. The current study examines regulation of binding sites by in vivo drug administration for adult, day 15, and day 5 rats. Drugs or saline were administered intraperitoneally, and striatal and cortical membrane preparations were assayed. Control [3H]hemicholinium-3 binding increases twofold between postnatal days 5 and 15 only in striatum. After day 15, binding increases 2.7-fold in cortex and striatum. Nicotine treatment increases striatal and cortical hemicholinium-3 binding at all three ages, with greater percent increases at day 5. Haloperidol increases binding only in striatum, again with larger effects at day 5. Both striatal and cortical binding are reduced by oxotremorine; however, the magnitude of this effect is unchanged during development. Pentobarbital reduces binding only in striatum, with no developmental change. Atropine and apomorphine do not change binding from control values. In summary, all drug treatments effective in adults were already effective by day 5. Cholinergic terminals present early in development are regulated by similar nicotinic and muscarinic cholinergic, dopaminergic, and sedative-hypnotic mechanisms as the adult. Changes in magnitude may be due to changes in drug metabolism or to developmental differences in regulation.     

Neural basis of learning new movements: evolution of classical concepts

According to classical consepts, the role of the motor cortex in performance of skilled movements of distal parts of extremities is confined to control of appropriate motoneurons by the "point-to-point" principle. However, much evidence of plasticity of the motor cortex and its active role in motor learning appeared in last decade. Fos-gene expression in the motor cortex was found to accompany learning a skill. Strengthening of horizontal pathways in layers II-III was revealed, and cholinergic input to tese layers was found to be important. The imaging data show that activity of the motor cortex increases during motor practice as well. This raises the question of specificity of the motor cortex in the motor learning per se. During acquisition of new movements some previously used synergies prevent the necessary coordination from being learned, so they must be suppressed in the process of motor learning. Investigations of central mechanisms of coordination interference in humans are still at the beginning. However, there are some animal models of reorganization and suppression of interfering synergies. The reorganization and suppression of coordination preventing realization of a new movement is shown to be a specific function of the motor cortex. After automation of new synergies the cortical control is still present, as distinct from the learned movements, which do not require suppression of interfering synergies. However, it does not mean that the conscious control of the performance is still present.     

Magnetic stimulation of the human motor cortex evokes skin sympathetic nerve activity.

Single-pulse magnetic coil stimulation (Cadwell MES 10) over the cranium induces without pain an electric pulse in the underlying cerebral cortex. Stimulation over the motor cortex can elicit a muscle twitch. In 10 subjects, we tested whether motor cortical stimulation could also elicit skin sympathetic nerve activity (SSNA; n = 8) and muscle sympathetic nerve activity (MSNA; n = 5) in the peroneal nerve. Focal motor cortical stimulation predictably elicited bursts of SSNA but not MSNA; with successive stimuli, the SSNA responses did not readily extinguish (94% of discharges to the motor cortex evoked SSNA responses) and had predictable latencies [739 +/- 33 (SE) to 895 +/- 13 ms]. The SSNA responses were similar after stimulation of dominant and nondominant sides. Focal stimulation posterior to the motor cortex elicited extinguishable SSNA responses. In three of six subjects, anterior cortical stimulation evoked SSNA responses similar to those seen with motor cortex stimulation but without detectable movement; in the other subjects, anterior stimulation evoked less SSNA discharge than that seen with motor cortex stimulation. Contrasting with motor cortical stimulation, evoked SSNA responses were more readily extinguished with 1) peripheral stimulation that directly elicited forearm muscle activation accompanied by electromyograms similar to those with motor cortical stimulation; 2) auditory stimulation by the click of the energized coil when off the head; and 3) in preliminary experiments, finger afferent stimulation sufficient to cause tingling. Our findings are consistent with the hypothesis that motor cortex stimulation can cause activation of both alpha-motoneurons and SSNA.     

Environmental control of collateral branching and target invasion of mitral cell axons during development

During development, mitral cell axons, the major efferents of the olfactory bulb, exhibit a protracted waiting period in the lateral olfactory tract (LOT) before giving off collateral branches and innervating the target olfactory cortex. To investigate the target invasion mechanism, a series of heterochronic and heterotopic cocultures of olfactory bulbs with various olfactory cortical strips were conducted. These experiments indicated that development of collateral branches is triggered by environmental cues but not by intrinsic mechanisms in mitral cells. The collateral-inducing cues are apparently different from the cues directing outgrowth of primary mitral cell axons. Coculture experiments also indicated that the target olfactory cortex undergoes a developmental change to become accessible to mitral cell fibers. Primary mitral cell axons, however, still preferred the LOT position over such accessible piriform cortex when encountered both the locations. These results suggest that mitral cell projection comprises multiple steps which are controlled by various environmental cues.