Evidence for a deficit in cholinergic interneurons in the striatum in schizophrenia

Cooperation between vision and somatomotor behavior, such as manual exploration of objects, suggests close functional coupling between the visual and sensorimotor systems. We observed this type of interaction in human volunteers during binocular rivalry while following the level of sensorimotor mu rhythm with a whole-scalp neuromagnetometer. The observers viewed a weak vertical grating in the lower visual field of one eye and a strong horizontal grating in the same spatial window of the other eye. When stationary, the weak grating was permanently invisible because of its low contrast and spatial frequency. A sudden brief drifting movement of the weak grating wiped out the dominant grating, and the weak grating became visible for less than the 3-s interval between the movements. The postcentral 8- to 15-Hz mu rhythm was found in six of nine observers, and its level increased transiently by 10-15%, starting about 450 ms after the beginning of the movement. The mu level was also enhanced by the actual disappearance of the stronger stimulus, when it occurred in random order with the rivalry stimuli. Identical visual motion, when not accompanied by a perceptual dominance change, produced only minor effects on the mu rhythm. Our results show that a change in visual percept, even with no real or imagined motor response, is associated with modified activity of the postcentral gyrus. This modification may reflect visuohaptic interactions and/or activity of the distributed cortical network implementing visually guided movements.     

NMDA and AMPA receptors contribute to the nicotinic cholinergic excitation of CA1 interneurons in the rat hippocampus

In the hippocampus, glutamatergic inputs to pyramidal neurons and interneurons are modulated by alpha7* and alpha3beta4* nicotinic acetylcholine receptors (nAChRs), respectively, present in glutamatergic neurons. This study examines how nicotinic AMPA, and NMDA receptor nAChR activities are integrated to regulate the excitability of CA1 stratum radiatum (SR) interneurons in rat hippocampal slices. At resting membrane potentials and in the presence of extracellular Mg2+ (1 mM), nicotinic agonists triggered in SR interneurons excitatory postsynaptic currents (EPSCs) that had two components: one mediated by AMPA receptors, and the other by NMDA receptors. As previously shown, nicotinic agonist-triggered EPSCs resulted from glutamate released by activation of alpha3beta4* nAChRs in glutamatergic neurons/fibers synapsing directly onto the neurons under study. The finding that CNQX caused more inhibition of nicotinic agonist-triggered EPSCs than expected from the blockade of postsynaptic AMPA receptors indicated that this nicotinic response also depended on the AMPA receptor activity in the glutamatergic neurons synapsing onto the interneuron under study. Nicotinic agonists always triggered action potentials in CA1 SR interneurons. In most interneurons, these action potentials resulted from activation of somatodendritic AMPA receptors and alpha7* nAChRs. In interneurons expressing somatodendritic alpha4beta2* nAChRs, activation of these receptors caused sufficient membrane depolarization to remove the Mg2+-induced block of somatodendritic NMDA receptors; in these neurons, nicotinic agonist-triggered action potentials were partially dependent on NMDA receptor activation. Removing extracellular Mg2+ or clamping the neuron at positive membrane potentials revealed the existence of a tonic NMDA current in SR interneurons that was unaffected by nAChR activation or inhibition. Thus integration of the activities of nAChRs, NMDA, and AMPA receptors in different compartments of CA1 neurons contributes to the excitability of CA1 SR interneurons.     

Postsynaptic targets of somatostatin-immunoreactive interneurons in the rat hippocampus

Two characteristic interneuron types in the hippocampus, the so-called hilar perforant path-associated cells in the dentate gyrus and stratum oriens/lacunosum-moleculare neurons in the CA3 and CA1 regions, were suggested to be involved in feedback circuits. In the present study, interneurons identical to these cell populations were visualized by somatostatin-immunostaining, then reconstructed, and processed for double-immunostaining and electron microscopy to establish their postsynaptic target selectivity. A combination of somatostatin-immunostaining with immunostaining for GABA or other interneuron markers revealed a quasi-random termination pattern. The vast majority of postsynaptic targets were GABA-negative dendritic shafts and spines of principal cells (76%), whereas other target elements contained GABA (8%). All of the examined neurochemically defined interneuron types (parvalbumin-, calretinin-, vasoactive intestinal polypeptide-, cholecystokinin-, substance P receptor-immunoreactive neurons) received innervation from somatostatin-positive boutons. Recent anatomical and electrophysiological data showed that the main excitatory inputs of somatostatin-positive interneurons originate from local principal cells. The present data revealed a massive GABAergic innervation of distal dendrites of local principal cells by these feedback driven neurons, which are proposed to control the efficacy and plasticity of entorhinal synaptic input as a function of local principal cell activity and synchrony.     

Evidence for the existence of galanin receptors on cultured astrocytes of rat CNS: colocalization with cholinergic receptors

The cellular localization of binding sites for [¹²⁵I]galanin was studied in explant cultures of rat neocortex, cerebellum, locus coeruleus and spinal cord by means of autoradiography. Binding sites for the peptide were observed on a great number of astrocytes in all CNS regions studied. In addition to astrocytes, many neurones were intensely labelled by [¹²⁵I]galanin. Binding of [¹²⁵I]galanin (10⁻⁸ M) to both astrocytes and neurones was markedly reduced or inhibited by the unlabelled peptide at high concentration (10⁻⁶ M), suggesting `specific' binding of the radioligand. Evidence for the colocalization of galanin and cholinergic receptors on astrocytes was provided by combined autoradiographic and immunohistochemical studies. Many astrocytes were labelled by [¹²⁵I]galanin and immunostained with antibodies to either muscarinic or nicotinic receptors. Electrophysiological studies revealed that addition of galanin (10⁻⁹ to 10⁻⁷ M) to the bathing fluid caused a dose-dependent hyperpolarization of the majority of astrocytes studied. When galanin (10⁻⁸ M) and the cholinergic agonists muscarine and nicotine (10⁻⁶ M) were tested on the same astrocyte, all three compounds induced a hyperpolarization, suggesting a colocalization of functional galanin and cholinergic receptors on the glial membrane.     

GABAergic circuits mediate the reinforcement-related signals of striatal cholinergic interneurons

Neostriatal cholinergic interneurons are believed to be important for reinforcement-mediated learning and response selection by signaling the occurrence and motivational value of behaviorally relevant stimuli through precisely timed multiphasic population responses. An important problem is to understand how these signals regulate the functioning of the neostriatum. Here we describe the synaptic organization of a previously unknown circuit that involves direct nicotinic excitation of several classes of GABAergic interneurons, including neuroptide Y-expressing neurogilaform neurons, and enables cholinergic interneurons to exert rapid inhibitory control of the activity of projection neurons. We also found that, in vivo, the dominant effect of an optogenetically reproduced pause-excitation population response of cholinergic interneurons was powerful and rapid inhibition of the firing of projection neurons that is coincident with synchronous cholinergic activation. These results reveal a previously unknown circuit mechanism that transmits reinforcement-related information of ChAT interneurons in the mouse neostriatal network.     

Age-related alterations in the rodent brain cholinergic system and behavior

Pre- and postsynaptic cholinergic markers were studied in various brain regions of mice and rats aged 6 to 30 months in an attempt to determine whether alterations in this transmitter system occur during the normal aging process. Reliable decreases in cholinergic receptor binding and choline acetyltransferase (CAT) activity were found in the cerebral cortex and corpus striatum. These alterations in the cholinergic system were typically more consistent and robust than changes involving glutamic acid decarboxylase, an enzyme marker for GABA neurons. No statistically significant changes in any markers were found in the hippocampus of either species. Significant age-related changes in retention of passive avoidance learning and locomotor activity were also observed in these same animals. These findings demonstrate that changes in the cholinergic system occur naturally in aged mice and rats and that both the loss of cholinergic receptors and decrease in CAT activity may contribute to the motor and mental impairments that often accompany old age.     

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [³H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [³H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A.

This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.     

Acetylcholine levels increase in rat hippocampus following acute septal lesions: Evidence for interactions between cholinergic and noncholinergic neurons

Acute septal lesions in rat brain resulted in elevation of the amount of particle-bound acetylcholine in the hippocampus irrespective of the extent of damage to the cholinergic septohippocampal projection. Changes in the high affinity choline uptake in the hippocampus were, however, proportional to the degree of destruction of this projection. The results are discussed in terms of possible interactions between the cholinergic and noncholinergic pathways in the system investigated.     

Postnatal development and migration of cholecystokinin-immunoreactive interneurons in rat hippocampus

The development of cholecystokinin-immunoreactive (CCK-IR) interneurons in the rat hippocampus was studied using immunocytochemical methods at the light and electron microscopic levels from early (P0-P8) to later postnatal (P12-P20) periods. The laminar distribution of CCK-IR cell bodies changed considerably during the studied period, which is suggested to be due to migration. CCK-IR cells appear to move from the molecular layer of the dentate gyrus to their final destination at the stratum granulosum/hilus border, and tend to concentrate in the distal third of stratum radiatum in CA1-3. The density of CCK-IR cells is rapidly decreasing during the first 4 postnatal days without any apparent reduction in their total number, therefore it is due to the pronounced growth of hippocampal volume in this period. Axons of CCK-IR interneurons formed symmetrical synapses already at P0, and by far the predominant targets were dendrites of presumed principal cells in all subfields of the hippocampus. These axon arbors began to concentrate around pyramidal cell bodies only at P8, at earlier ages CCK-IR axons crossed stratum pyramidale at right angles, and gave rise to varicose collaterals only outside this layer. The dendrites and somata of CCK-IR cells received synapses already at P0, but those were mostly symmetrical, apart from a few immature asymmetrical synapses. At P4, mature asymmetrical synapses with considerable amounts of synaptic vesicles were already commonly encountered. Thus, the innervation of CCK-IR interneurons apparently develops later than their output synapses, suggesting that they may be able to release transmitter before receiving any considerable excitatory drive. We conclude that CCK-IR cells represent one, if not the major, interneuron type that assists in the maturation of glutamatergic synapses (activation of N-methyl-D-aspartate receptors) via GABAergic depolarization of principal cell dendrites, and may contribute to the generation of giant depolarizing potentials. CCK-IR cells will change their function to perisomatic hyperpolarizing inhibition, as glutamatergic transmission in the network becomes operational.     

Origin of the slow afterhyperpolarization and slow rhythmic bursting in striatal cholinergic interneurons

Striatal cholinergic interneurons recorded in slices exhibit three different firing patterns: rhythmic single spiking, irregular bursting, and rhythmic bursting. The rhythmic single-spiking pattern is governed mainly by a prominent brief afterhyperpolarization (mAHP) that follows single spikes. The mAHP arises from an apamin-sensitive calcium-dependent potassium current. A slower AHP (sAHP), also present in these neurons, becomes prominent during rhythmic bursting or driven firing. Although not apamin sensitive, the sAHP is caused by a calcium-dependent potassium conductance. It is not present after blockade of calcium current with cadmium or after calcium is removed from the media or when intracellular calcium is buffered with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. It reverses at the potassium equilibrium potential. It can be generated by subthreshold depolarizations and persists after blockade of sodium currents by tetrodotoxin. It is slow, being maximal approximately 1 s after depolarization onset, and takes several seconds to decay. It requires >300-ms depolarizations to become maximally activated. Its voltage sensitivity is sigmoidal, with a half activation voltage of -40 mV. We conclude the sAHP is a high-affinity apamin-insensitive calcium-dependent potassium conductance, triggered by calcium currents partly activated at subthreshold levels. In combination with those calcium currents, it accounts for the slow oscillations seen in a subset of cholinergic interneurons exhibiting rhythmic bursting. In all cholinergic interneurons, it contributes to the slowdown or pause in firing that follows driven activity or prolonged subthreshold depolarizations and is therefore a candidate mechanism for the pause response observed in cholinergic neurons in vivo.     

Evidence for existence of ST-16 specificity in Chinese

The two new HLA-B specificities ST-16 and SH6 were defined for the first time with the local sera in Mexican-Americans and Chinese, respectively. Using the 9th Workshop sera, it was confirmed that SH6, Bw6-associated split of B16, was serologically identical to ST-16.     

Synaptic signaling between GABAergic interneurons and oligodendrocyte precursor cells in the hippocampus

Oligodendrocyte precursor cells (OPCs) express receptors for many neurotransmitters, but the mechanisms responsible for their activation are poorly understood. We have found that quantal release of GABA from interneurons elicits GABA(A) receptor currents with rapid rise times in hippocampal OPCs. These currents did not exhibit properties of spillover transmission or release by transporters, and immunofluorescence and electron microscopy suggest that interneuronal terminals are in direct contact with OPCs, indicating that these GABA currents are generated at direct interneuron-OPC synapses. The reversal potential of OPC GABA(A) currents was -43 mV, and interneuronal firing was correlated with transient depolarizations induced by GABA(A) receptors; however, GABA application induced a transient inhibition of currents mediated by AMPA receptors in OPCs. These results indicate that OPCs are a direct target of interneuronal collaterals and that the GABA-induced Cl(-) flux generated by these events may influence oligodendrocyte development by regulating the efficacy of glutamatergic signaling in OPCs.     

Strychnine-sensitive glycine receptors in rat caudatoputamen are expressed by cholinergic interneurons

Strychnine-sensitive glycine receptors are ligand-gated anion channels widely expressed in spinal cord and brainstem. Recent functional studies demonstrating glycine-induced release of [(3)H]acetylcholine in rat caudatoputamen suggested the existence of excitatory glycine receptors in that region. Since the expression of glycine receptors in the caudatoputamen had not been reported earlier, we studied the glycine receptor-like immunoreactivity in this structure using a monoclonal antibody (mAb4a) recognizing an epitope common to all of the ligand-binding alpha-subunit variants of the glycine receptor. [Becker et al. (1993) Brain Res. 11, 327-333; Nicola et al. (1992) Neurosci. Lett. 138, 173-178]. Immunohistochemistry with mAb4a disclosed a specific staining of sparsely distributed large neurons in rat caudatoputamen, displaying an immunoreactive signal of lower intensity than that observed in motoneurons in spinal cord. Fluorescent dual labelling demonstrated that glycine receptor-like immunoreactivity co-localizes with choline acetyltransferase-like immunoreactivity in rat caudatoputamen. All neurons with glycine receptor-like immunoreactivity in the caudatoputamen studied were immunoreactive with choline acetyltransferase, and represented a subpopulation of cholinergic neurons (approximately 90% of the somata with choline acetyltransferase-like immunoreactivity).These results suggest that strychnine-sensitive glycine receptors are present on cholinergic interneurons in rat caudatoputamen, supporting the hypothesis that glycine receptors inducing striatal release of [(3)H]acetylcholine may be localized to cholinergic neurons.     

大鼠杏仁体基底外侧核GABA和ACh能中间神经元的突触

目的：揭示杏仁体基底外侧核(BL)中的γ-氨基丁酸(GABA)能和乙酰胆碱能(ACh)2种中间神经元树突上的突触联系。方法：用抗GABA和抗胆碱乙酰转移酶(ChAT)抗体对BL做光镜和电镜免疫组化染色。结果：光镜下,GABA免疫反应阳性神经元多为圆形多极神经元;CHAT免疫阳性神经元多为双极神经元。两者数量比约为(7-9)：1。电镜下,支配GABA免疫阳性神经元的突触52．7％为非对称性(兴奋性),47．3％为对称性(抑制性),而ChAT免疫阳性神经元则分别为44．9％和55．1％。结论：在情绪性学习记忆的处理过程中,杏仁体GABA能中间神经元起主要的功能作用,可能为抑制作用;而ACh能中间神经元作为辅助的均衡调节作用。     

Accumbens cholinergic interneurons play a role in the regulation of body weight and metabolism

The aims of the present study were (1) to determine whether selective lesions of the accumbens cholinergic interneurons impair feeding and body weight regulation, and (2) to characterize the nature of disturbances using motivational and metabolic challenges. Rats with bilateral cholinotoxic (AF64A) lesions in the nucleus accumbens showed a significant and lasting lag in body weight gain in comparison to the sham-operated controls. This failure to gain weight was not due to a decrease in feeding because lesioned rats actually ate more food and drank more water than controls under basal conditions. Lesion-induced deficits were also exposed when the rats were challenged with food deprivation or cold exposure. Lesioned rats ate less than controls when 24 h food deprived and maintained both a higher core temperature and a higher metabolic rate than controls following either 24-h food deprivation or exposure to cold. Thyroid hormones, insulin, and blood glucose levels were, however, within the physiological range, and no sensory and motor disturbances were observed. The results suggest that the altered body weight regulation is partly due to the enhanced metabolic responsiveness to stress. Possible explanations for the effects of the lesions are also discussed in the context of motivational alterations, including possible dopamine-acetylcholine interactions.     

Muscarinic cholinergic receptor subtypes in the hippocampus of aged rats.

In spite of the suggestion of impaired muscarinic function in adult-onset cognitive disorders, data on the expression of muscarinic receptors in the hippocampus as a function of age are inconsistent. One reason may be that the majority of investigations were unable to differentiate the five brain muscarinic receptors subtypes. In this study, using a protocol based on a combination of both kinetic and equilibrium binding approaches, we have assessed the expression and the density of M1-M5 muscarinic cholinergic receptors in the hippocampus of Fisher 344 rats aged 6, 15 and 22 months. An age-related decrease of the density of M1 receptor was found in pyramidal neurons of the CA1 subfield. In this area, other subtypes of muscarinic receptors were unchanged with the exception of a loss of M2 receptor in the radial layer. In the CA3 subfield, receptor changes involved M2, M3 and M5 subtypes, whereas in the dentate gyrus, the main changes affected M1 and M2 receptors of the granular layer and M2 and M3 receptors of the molecular layer. The above findings indicate that analysis of age-related changes of different muscarinic cholinergic receptors might represent a useful contribution to identifying the basis of cholinergic neurotransmission impairment in adult-onset cognitive dysfunction.     

Comparative immunohistochemistry of synaptic markers in the rodent hippocampus in pilocarpine epilepsy

Pilocarpine-induced epileptic state (Status epilepticus) generates an aberrant sprouting of hippocampal mossy fibers, which alter the intrahippocampal circuits. The mechanisms of the synaptic plasticity remain to be determined. In our studies in mice and rats, pilocarpine-induced seizures were done in order to gain information on the process of synaptogenesis. After a 2-month survival period, changes in the levels of synaptic markers (GAP-43 and Syn-I) were examined in the hippocampus by means of semi-quantitative immunohistochemistry. Mossy fiber sprouting (MFS) was examined in each brain using Timm's sulphide-silver method. Despite the marked behavioral manifestations caused by pilocarpine treatment, only 40% of the rats and 56% of the mice showed MFS. Pilocarpine treatment significantly reduced the GAP-43 immunoreactivity in the inner molecular layer in both species, with some minor differences in the staining pattern. Syn-I immunohistochemistry revealed species differences in the sprouting process. The strong immunoreactive band of the inner molecular layer in rats corresponded to the Timm-positive ectopic mossy fibers. The staining intensity in this layer, representing the ectopic mossy fibers, was weak in the mouse. The Syn-I immunoreactivity decreased significantly in the hilum, where Timm's method also demonstrated enhanced sprouting. This proved that, while sprouted axons displayed strong Syn-I staining in rats, ectopic mossy fibers in mice did not express this synaptic marker. The species variability in the expression of synaptic markers in sprouted axons following pilocarpine treatment indicated different synaptic mechanisms of epileptogenesis.