No abstract

The existence of cholinergic neuronal cell bodies in mammalian cerebral cortex was long the subject of much controversy (see ref. 1 for review). Recently, however, a specific cholinergic marker, the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT, E.C.2.3.1.6), was demonstrated by immunohistochemical methods to be present in bipolar neurones in rat cortex. Here we show that at least 80% of these intrinsic cholinergic neurones also contain immunoreactivity for vasoactive intestinal polypeptide (VIP), a neuroactive peptide found to be present in a subpopulation of cortical neurones. On the other hand, we find that the ChAT-positive cells in the basal forebrain, which are another major source of cholinergic innervation of the cortex, contain no detectable VIP-immunoreactivity. In addition, we have observed by both light and electron microscopy that some VIP- and some ChAT-positive structures in cortex are closely associated with blood vessels.

Simultaneous whole cell recordings from monosynaptically connected cortical cells were performed with the use of two patch pipettes to determine the effect of acetylcholine (ACh) on both excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs, respectively) in cultured neurons from rat visual cortex. For 96% of EPSPs and 73% of IPSPs, ACh potently suppressed postsynaptic potentials in a dose-dependent manner. The estimated effective concentrations to produce half maximal response (EC50S) were 30 and 210 nM for EPSPs and IPSPs, respectively. To identify what subtypes of ACh receptors are involved in the suppression of postsynaptic potentials, three different, partially selective muscarinic receptor antagonists were used. According to the comparison of estimated Schild coefficients for each of the three antagonists against the suppression by ACh, EPSPs are most likely mediated by m4 receptors, and IPSPs by m1 receptors. When cells were treated with pertussis toxin, which inactivates m2 and m4 receptors while leaving m1, m3, and m5 receptors intact, 7 of 8 EPSPs were resistant to ACh whereas 8 of 12 IPSPs were still suppressed by ACh. This result supports the interpretation that the suppression of EPSPs was mediated by m4 receptors and that of IPSPs by m1 receptors. To obtain an indication as to whether ACh works presynaptically or postsynaptically, 1/CV2 analysis was carried out. The resultant diagonal alignment of the ratio of 1/CV2 plotted against the ratio of the amplitude of postsynaptic potentials suggests a presynaptic mechanism for the suppression of both EPSPs and IPSPs. In addition, in many cases a large synaptic suppression was observed without an obvious change in the input resistance. Furthermore, in one case where a single inhibitory driver cell was recorded with three different follower cells sequentially, none of the three IPSPs was suppressed by ACh, providing additional support for the presynaptic localization of ACh action. These results suggest that in cerebral cortex ACh has, in addition to its direct facilitatory effect via m3 pharmacology, a suppressive effect on EPSPs and IPSPs via m1 and m4 muscarinic receptors, respectively, probably with a presynaptic site of action. Separation of the actions of ACh into different receptor-second messenger pathways with potential for independent interactions with other neuromodulatory systems may be an important aspect of the mechanism of cholinergic regulation of functional state in cortex. Separation of cholinergic effects at different receptors might also offer a means for selective pharmacological intervention in disorders of sleep or memory.

Acetylcholine and dopamine are key neurotransmitters in the extrapyramidal motor system, where they are thought to lie in a 'functional balance' brought about by interactions between the terminals of the dopamine-containing nigrostriatal tract and the cholinergic interneurones of the striatum. The precise nature of these interactions is not understood, however, nor is it clear how they influence the functioning of striatal systems containing other neurotransmitters. A new clue to understanding such interplay among transmitter-coded systems in the striatum has come from the finding that many of them, including nigrostriatal afferents, follow a macroscopic ordering in which neural elements are concentrated either in or out of the striatal tissue compartments called striosomes. We here report that the cholinergic neuropil of the striatum is also compartmentalized: fibres expressing immunoreactivity to antibodies raised against choline acetyltransferase (ChAT) are sparse in striosomes and are dense in the extrastriosomal matrix. These findings suggest that the interactions between acetylcholine and other neurotransmitters in the striatum are spatially constrained, that cholinergic modulation of striatal function predomintes in the extrastriosomal matrix, and that extrapyramidal pathways originating in the matrix, including transthalamic pathways to the frontal lobes, may in particular reflect this cholinergic influence. Such a differential organization of striatal cholinergic circuitry could help to account for the selective therapeutic efficacy of anticholinergic drugs in the treatment of extrapyramidal disorders.

The vestibular primary afferent projection to the cerebellum of the rabbit was studied with retrograde and orthograde tracers. We injected individual lobules of the cerebellum with horseradish peroxidase (HRP) or wheat germ agglutinin-HRP (WGA-HRP). Following these injections the numbers of labeled and unlabeled cells in Scarpa's ganglion were counted. Approximately 64-89% of the cells in Scarpa's ganglion were labeled retrogradely following uvula-nodular injections. About 2% of the cells in the ipsilateral Scarpa's ganglion were labeled after injections of the flocculus. Virtually no cells were labeled following injections of the ventral paraflocculus. The vestibular primary afferent projection to the uvula-nodulus is so extensive that it must be part of a collateral system that also innervates the vestibular nuclei. This collateral projection pattern was confirmed by using fluorescent tracers injected into the uvula-nodulus and vestibular complex. Fluorogold was injected into the uvula-nodulus and peroxidase-rhodamine isothiocyanate was injected into the vestibular complex. More than 50% of the neurons in Scarpa's ganglion were double labeled by these subtotal injections. The dense vestibular primary afferent projection to the uvula-nodulus was confirmed by using the C fragment of tetanus toxin (TTC) injected into the labyrinth as an orthograde tracer. With the TTC technique, the vestibular primary afferent projection to the uvula-nodulus terminated exclusively in the ipsilateral granule cell layer of lobules 9d and 10. Much sparser vestibular primary afferent projections were found in the banks of major cerebellar sulci. A barely detectable projection was found to the flocculus and ventral paraflocculus.

The pharmacological properties of excitatory synapses on pyramidal cells in layer V of rat visual cortex were investigated by recording EPSPs intracellularly in tissue slices. The EPSPs were evoked by electrically stimulating cells in layer II/III or axons in white matter. All of the layer V neurons were pyramidal in nature as determined by injections of Lucifer yellow or by electrophysiological criteria. Application of the broadly acting antagonists kynurenic acid and gamma-D-glutamylglycine reversibly antagonized the EPSPs from both presynaptic sources in a dose-dependent manner: 1 and 5 mM kynurenic acid produced 63 and 79% reductions, respectively, of control responses. The specific NMDA antagonist APV (50 microM) caused a small reduction in peak amplitude and a more significant reduction in the duration of the falling phase of EPSPs. When slices were bathed in Mg2+-free medium, the amplitude of the EPSP increased substantially. Under these conditions APV reduced the size of the EPSP to that observed with APV in the presence of 1 mM Mg2+. The voltage sensitivities of the APV-sensitive and APV-insensitive components of the layer II/III-evoked EPSPs were examined. The APV-insensitive component was not voltage dependent and had an extrapolated reversal potential of -10 mV. In contrast, the APV-sensitive component showed an NMDA-like voltage dependency; it was greatest at the most positive potentials tested (-45 mV) and nearly absent at membrane potentials below rest. At potentials near threshold, the APV-sensitive component contributed approximately half of the total response. Although the time to peak and decay were longer for the APV-sensitive component, the latency was the same as that of the APV-insensitive component. These results provide evidence that the layer II/III to V pathway, which comprises a major interlaminar circuit in cortex, is mediated directly through NMDA as well as non-NMDA receptors located on the layer V cells. This finding has implications for the role of this circuit in cortical visual plasticity.

The cholinergic innervation of the cerebellar cortex of the rat, rabbit, cat and monkey was studied by immunohistochemical localization of choline acetyltransferase (ChAT) and radiochemical measurement of regional differences in ChAT activity. Four antibodies to ChAT were used to find optimal immunohistochemical localization of this enzyme. These antibodies selectively labeled large mossy fiber rosettes as well as finely beaded terminals with different morphological characterization, laminar distribution within the cerebellar cortex, and regional differences within the cerebellum. Large "grape-like" classic ChAT-positive mossy fiber rosettes that were distributed primarily in the granule cell layer were concentrated, but not exclusively located in three separate regions of the cerebellum in each of the four species studied: 1) The uvula-nodulus (lobules 9 and 10); 2) the flocculus-ventral paraflocculus, and 3) the anterior lobe vermis (lobules 1 and 2). No intrinsic cerebellar neurons were labeled. No cells in either the inferior olive (the origin of cerebellar climbing fibers) or in the locus coeruleus (an origin of noradrenergic fibers) were ChAT-positive. Thin, finely beaded axons, similar to cholinergic axons of the cerebral cortex of the rat, were observed in both the granule cell layer and molecular layer of the cerebellar cortex of the rat, rabbit and cat. The regional differences in ChAT-positive afferent terminations in the cerebellar cortex was for the most part confirmed by regional measurements of ChAT activity in the rat, rabbit, and cat. The three cholinergic afferent projection sites correspond to regions of the cerebellar cortex that receive vestibular primary and secondary afferents. These data imply that a subset of vestibular projections to the cerebellar cortex are cholinergic.

Previously we have shown that four regions of the cerebellum, the uvula-nodulus, flocculus, ventral paraflocculus, and anterior lobe 1, receive extensive, but not exclusive, cholinergic mossy fiber projections. In the present experiment we have studied the origin of three of these projections in the rat and rabbit (uvula-nodulus, flocculus, ventral paraflocculus), using choline acetyltransferase (ChAT) immunohistochemistry in combination with a double label, retrogradely transported horseradish peroxidase (HRP). We have demonstrated that in both the rat and rabbit the caudal medial vestibular nucleus (MVN) and to a lesser extent the nucleus prepositus hypoglossus (NPH) contain ChAT-positive neurons. Neurons of the caudal MVN are double-labeled following HRP injections into the uvula-nodulus. HRP injections into the uvula-nodulus also labeled less than 5% of the neurons in the cholinergic vestibular efferent complex. Fewer ChAT-positive neurons in the MVN and some ChAT-positive neurons in the NPH are double-labeled following HRP injections into the flocculus. Almost no ChAT-positive neurons in the MVN and some ChAT-positive neurons in the NPH are double-labeled following HRP injections into the ventral paraflocculus. Injections of Phaseolus leucoagglutinin (PHA-L) into the caudal MVN of both the rat and rabbit demonstrated projection patterns to the uvula-nodulus and flocculus that were qualitatively similar to those observed using ChAT immunohistochemistry. We conclude that the cholinergic mossy fiber pathway to the cerebellum in general and the uvula-nodulus in particular is likely to mediate secondary vestibular information related to postural adjustments.