Cholinergic innervation of the cat striate cortex: a choline acetyltransferase immunocytochemical analysis

In area 17 of adult cats the morphology, distribution, and synaptology of cholinergic elements were examined by immunocytochemical methods with a monoclonal antibody against choline acetyltransferase (ChAT). ChAT(+) fibers are present throughout the entire depth of the cortex but are particularly dense in layer I. Typically these fibers are very thin and possess numerous irregularly spaced varicosities. Except in layer I and deep layer VI, where the fibers tend to run parallel to the pial surface, they appear to be randomly oriented. At the electron microscope level, immunolabeling was present in unmyelinated fibers of irregular contour and diameter. Most of the ChAT(+) varicose profiles contained mitochondria and round vesicles. Synaptic complexes were relatively infrequent and tended to be of the symmetrical type. They were located mostly on dendritic shafts and only rarely on cell bodies and dendritic spines. Both pyramidal and nonpyramidal cells were found to be innervated by cholinergic afferents. These anatomical data are consistent with the known physiology of acetylcholine in the visual cortex, which indicates that it acts as a modulator of cortical excitability.     

Distribution of GABAergic neurons and axon terminals in the macaque striate cortex

Antisera to glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) have been used to characterize the morphology and distribution of presumed GABAergic neurons and axon terminals within the macaque striate cortex. Despite some differences in the relative sensitivity of these antisera for detecting cell bodies and terminals, the overall patterns of labeling appear quite similar. GABAergic axon terminals are particularly prominent in zones known to receive the bulk of the projections from the lateral geniculate nucleus; laminae 4C, 4A, and the cytochrome-rich patches of lamina 3. In lamina 4A, GABAergic terminals are distributed in a honeycomb pattern which appears to match closely the spatial pattern of geniculate terminations in this region. Quantitative analysis of axon terminals that contain flat vesicles and form symmetric synaptic contacts (FS terminals) in lamina 4C beta and in lamina 5 suggest that the prominence of GAD and GABA axon terminal labeling in the geniculate recipient zones is due, at least in part, to the presence of larger GABAergic axon terminals in these regions. GABAergic cell bodies and their initial dendritic segments display morphological features characteristic of nonpyramidal neurons and are found in all layers of striate cortex. The density of GAD and GABA immunoreactive neurons is greatest in laminae 2-3A, 4A, and 4C beta. The distribution of GABAergic neurons within lamina 3 does not appear to be correlated with the patchy distribution of cytochrome oxidase in this region; i.e., there is no significant difference in the density of GAD and GABA immunoreactive neurons in cytochrome-rich and cytochrome-poor regions of lamina 3. Counts of labeled and unlabeled neurons indicate that GABA immunoreactive neurons make up at least 15% of the neurons in striate cortex. Layer 1 is distinct from the other cortical layers by virtue of its high percentage (77-81%) of GABAergic neurons. Among the other layers, the proportion of GABAergic neurons varies from roughly 20% in laminae 2-3A to 12% in laminae 5 and 6. Finally, there are conspicuous laminar differences in the size and dendritic arrangement of GAD and GABA immunoreactive neurons. Lamina 4C alpha and lamina 6 are distinguished from the other layers by the presence of populations of large GABAergic neurons, some of which have horizontally spreading dendritic processes. GABAergic neurons within the superficial layers are significantly smaller and the majority appear to have vertically oriented dendritic processes.(ABSTRACT TRUNCATED AT 400 WORDS)     

An investigation of cholinergic circuitry in cat striate cortex using acetylcholinesterase histochemistry

The organization of cholinergic inputs to cat striate cortex (area 17) was studied by using a histochemical stain for acetylcholinesterase (AChE). Axons were labelled in all layers of the striate cortex, with distinct plexuses occurring in layer I, lower layer III, layer IVc, and layer VI. In addition to the stained axons, a population of layer V pyramidal cells was intensely AChE-positive. Surgical undercutting eliminated virtually all of the AChE-positive axons in the striate cortex, thus indicating that this innervation arises entirely from an extrinsic source in the cat. To identify this source, cell groups projecting to area 17 were retrogradely labelled with horseradish peroxidase. Cell groups labelled with horseradish peroxidase that were also intensely AChE-positive were considered as possible candidates for providing the cholinergic input to the striate cortex. These included the basal forebrain, several intralaminar nuclei, and the lateral geniculate nucleus. Kainate lesions were then made in each of these structures to assess their individual contributions to the cortical AChE pattern. Cortical AChE was depleted only after lesions of the basal forebrain, suggesting that this is the sole source of AChE-positive axons in area 17. Because the cortically projecting cells in this region have been shown to contain choline acetyltransferase in a number of species, we postulate that the AChE-positive fibers we describe in the cat striate cortex are in fact cholinergic.     

Evidence for interlaminar inhibitory circuits in the striate cortex of the cat

An interlaminar, ascending, and GABAergic projection is demonstrated in the striate cortex of the cat. We have examined a basket cell, with soma and smooth dendrites in layers V and VI, that was injected intracellularly with HRP in the kitten. Three-dimensional reconstruction of its axon revealed a horizontal plexus in layer V and upper VI, extending about 1.8 mm anteroposteriorly and 0.8 mm mediolaterally; a dense termination in the vicinity of the soma in layers V and VI; and an ascending tuft terminating in layers II and III in register above the soma and about 250 microns in diameter. Many boutons of this cell contacted neuronal somata and apical dendrites of pyramidal cells and subsequent electron microscopy showed that these boutons formed type II synaptic contacts with these structures. A random sample of postsynaptic targets (n = 199) in layers III, V, and VI showed that somata (20.1%), dendritic shafts (38.2%), and dendritic spines (41.2%) were contacted. The fine structural characteristics of postsynaptic elements indicated that the majority originated from pyramidal cells. Direct identification of postsynaptic neurons was achieved by Golgi impregnation of four large pyramidal cells in layer V, which were contacted on their somata and apical dendrites by between three and 34 boutons of the HRP-filled basket cell. Layer IV neurons were not contacted. Golgi-impregnated neurons similar to the HRP-filled basket cell were also found in the deep layers. The axonal boutons of one of them were studied; it also formed type II synapses with somata and apical dendrites of pyramidal cells. Boutons of the HRP-filled neuron were shown to be GABA-immunoreactive by the immunogold method. This is direct evidence in favour of the GABAergic nature of deep layer basket cells with ascending projections. The existence of an ascending GABAergic pathway was also demonstrated by injecting [3H]GABA into layers II and III. The labelled amino acid was transported retrogradely by a subpopulation of GABA-immunoreactive cells in layers V and VI, in addition to cells around the injection site. The axonal pattern and mode of termination of deep basket cells make them a candidate for producing or enhancing directional selectivity, a characteristic of layer V cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of choline acetyltransferase immunoreactive axons and terminals in the rat and ferret brainstem.

A survey was made of the density of the cholinergic innervation of different parts of the brainstem of the rat and ferret. Sections of rat and ferret brainstems were stained for choline acetyltransferase (ChAT) immunoreactivity by using a sensitive immunocytochemical method. Adjacent sections were stained for acetylcholinesterase activity or Nissl substance. The density of the distribution of fine calibre, varicose ChAT-positive axons, assumed to represent cholinergic terminals, was categorised arbitrarily into high, medium, or low. A high density of ChAT-positive terminals was found in all or parts of these structures: interpeduncular nucleus, superficial grey layer of the superior colliculus (ferret), intermediate layers of the superior colliculus, lateral part of the central grey (rat), an area medial to the parabigeminal nucleus (rat), pontine nuclei, ventral tegmental nucleus (rat), midline pontine reticular formation, and an area ventral to the exit point of the 5th nerve (ferret). A medium density of ChAT-positive terminals was observed in all or parts of: the substantia nigra zona compacta (ferret), ventral tegmental area (ferret), superficial grey layer of the superior colliculus, intermediate and deep layers of the superior colliculus, lateral central grey, area medial to the parabigeminal nucleus, inferior colliculus, dorsal tegmental nucleus, ventral tegmental nucleus (ferret), pontine nuclei, ventral nucleus of the lateral lemniscus (ferret), midline pontine reticular formation, ventral cochlear nucleus, dorsal cochlear nucleus, lateral superior olive, spinal trigeminal nuclei, prepositus hypoglossal nucleus, lateral reticular nucleus, paragigantocellular nucleus, and the dorsal column nuclei including the cuneate, external cuneate, and gracile nuclei. A low density of ChAT-positive terminals was seen throughout the remainder of the brainstem of the rat and ferret, but these terminals were absent from the medial superior olive, substantia nigra zona reticulata (rat), and the central part of the ferret lateral superior olive. A pericellular-like distribution of ChAT-positive terminals was observed in the ventral cochlear nucleus and in association with some of the cells of the nucleus of the mesencephalic tract of the trigeminal nerve. A climbing fibre type arrangement of ChAT-positive terminals was found in the substantia nigra zona compacta (ferret) and medial reticular formation. In general, the distribution of staining for AChE activity reflected that of the distribution of ChAT immunoreactivity in the brainstem, except in a few regions where there were also species differences in the distribution of ChAT-positive terminals, e.g., in the superficial grey layer of the superior colliculus and in the substantia nigra.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses

Choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme and a definitive marker for cholinergic neurons, was localized immunocytochemically in the motor and somatic sensory regions of rat cerebral cortex with monoclonal antibodies. ChAT-positive (ChAT+) varicose fibers and terminal-like structures were distributed in a loose network throughout the cortex. Some immunoreactive cortical fibers were continuous with those in the white matter underlying the cortex, and many of these fibers presumably originated from subcortical cholinergic neurons. ChAT+ fibers appeared to be rather evenly distributed throughout all layers of the motor cortex, but a subtle laminar pattern was evident in the somatic sensory cortex, where lower concentrations of fibers in layer IV contrasted with higher concentrations in layer V. Electron microscopy demonstrated that immunoreaction product was concentrated in synaptic vesicle-filled profiles and that many of these structures formed synaptic contacts. ChAT+ synapses were present in all cortical layers, and the majority were of the symmetric type, although a few asymmetric ones were also observed. The most common postsynaptic elements were small to medium-sized dendritic shafts of unidentified origin. In addition, ChAT+ terminals formed synaptic contacts with apical and, probably, basilar dendrites of pyramidal neurons, as well as with the somata of ChAT-negative nonpyramidal neurons. ChAT+ cell bodies were present throughout cortical layers II-VI, but were most concentrated in layers II-III. The somata were small in size, and the majority of ChAT+ neurons were bipolar in form, displaying vertically oriented dendrites that often extended across several cortical layers. Electron microscopy confirmed the presence of immunoreaction product within the cytoplasm of small neurons and revealed that they received both symmetric and asymmetric synapses on their somata and proximal dendrites. These observations support an identification of ChAT+ cells as nonpyramidal intrinsic neurons and thus indicate that there is an intrinsic source of cholinergic innervation of the rat cerebral cortex, as well as the previously described extrinsic sources.     

Distribution of GABAergic synaptic terminals on the dendrites of locust spiking local interneurones

Double-labelling and electron microscopy were used to assess the distribution of GABAergic synapses made onto the neurites of spiking local interneurones in the locust. The aims were to determine the sites of inputs mediating inhibition of the spiking local interneurones and to ascertain the relative abundance of such inputs. This information should allow us to understand better the integrative properties of these spiking local interneurones and the role of inhibition in shaping their receptive field properties or in fine tuning their spike-mediated outputs. Spiking interneurones in a midline population were labelled by intracellular injection of horseradish peroxidase after physiological characterisation. Colloidal gold immunocytochemistry was then used on ultrathin sections of these neurones with a polyclonal antibody raised against GABA. Most GABAergic (inhibitory) input synapses onto the interneurones are made on their ventral neurites, which also receive afferent (excitatory) inputs. These inhibitory inputs to the ventral neurites constitute 43% of the identifiable synapses. Relatively few GABAergic inputs were found onto the dorsal neurites, which are predominantly the sites of output synapses from these interneurones. These results suggest that much synaptic integration takes place in the ventral field of branches and that GABA-mediated presynaptic inhibitory control of spike-mediated outputs from the dorsal neurites is unlikely to occur. © 1993 Wiley-Liss, Inc.     

Decreased proportion of GABA neurons accompanies age-related degradation of neuronal function in cat striate cortex

Electrophysiological studies indicate that a decline of GABAergic inhibition in the visual cortex may underlie age-related degradation of visual function [A.G. Leventhal, Y. Wang, M. Pu, Y. Zhou, Y. Ma, GABA and its agonists improved visual cortical function in senescent monkeys, Science 300 (2003) 812-815; M.T. Schmolesky, Y. Wang, M. Pu, A.G. Leventhal, Degradation of stimulus selectivity of visual cortical cells in senescent rhesus monkeys, Nat. Neurosci. 3 (2000) 384-390]. To date, there is little direct evidence to support this hypothesis. Using Nissl staining and immunohistochemical techniques, we quantitatively compared the density of total neurons (Nissl-stained neurons) and GABA-immunoreactive neurons as well as the proportion of GABA-immunoreactive neurons to total neurons in the primary visual cortex between 4 young adult (1-3 year old) cats and 4 old (12 year old) cats, which had been previously examined in a single-unit recording study [T. Hua, X. Li, L. He, Y. Zhou, Y. Wang, A.G. Leventhal, Functional degradation of visual cortical cells in old cats, Neurobiol. Aging 27 (2006) 155-162]. In that study, we found the function of V(1) (area 17) neurons in the old cats was significantly degraded relative to young adult cats. Our present results indicate that the density of total neurons in each cortical layer of V(1) exhibit no significant difference in the two age groups of cats. However, the density of GABA-immunoreactive neurons in old cats is significantly lower than in young adults. Further, the ratio of GABA-immunoreactive neurons to total neurons in each layer of V(1) in old cats is also significantly decreased when compared to young adult cats. These results provide direct morphological evidence of decreased GABAergic inhibition in the striate visual cortex of old animals, which accompany the functional degradation of visual cortical neurons.     

Arborisation pattern and postsynaptic targets of physiologically identified thalamocortical afferents in striate cortex of the macaque monkey

The monosynaptic targets of different functional types of geniculocortical axons were compared in the primary visual cortex of monkeys. Single thalamocortical axons were recorded extracellularly in the white matter by using horseradish-peroxidase-filled pipettes. Their receptive fields were mapped and classified as corresponding to those of parvi- or magnocellular neurons in the lateral geniculate nucleus. The axons were then impaled and injected intraaxonally with horseradish peroxidase. Two magnocellular (MA) and two parvicellular (PA) axons were successfully recovered and reconstructed in three dimensions. The two MA axons arborised mainly in layer 4C alpha, as did the two PA axons in layer 4C beta. Few collaterals formed varicosities in layer 6. Both MA axons had two large, elongated clumps of bouton (approx. 300-500 x 600-1,200 microns each) and a small clump. One PA axon had two clumps (each with a core appr. 200 microns in diameter); the other had only one (appr. 150-200 microns in axon had 1,380; one MA axon had 3,200 boutons; and those of the more extensive MA axon were not counted. The distribution of postsynaptic targets as well as the number of synapses per bouton has been established for a sample of 150 PA boutons and 173 MA boutons from serial ultrathin sections. The MA axons made on average 2.1 synapses per bouton compared to 1.79 for one PA axon and 2.6 for the other. The sample of boutons taken from the two physiological types of axons contacted similar proportions of dendritic spines (52-68%), shafts (33-47%), and somata (0-3%). The postsynaptic elements were further characterized by immunostaining for GABA. All postsynaptic perikarya and some of the dendrites (4.5-9.5% of all targets) were positive for the amino acid. Near the thalamic synapse GABA-negative dendritic shafts frequently contained lamellar bodies, an organelle identical in structure to spine apparatus. Dendritic shafts and spines postsynaptic to the thalamocortical boutons frequently received an adjacent synapse from GABA-immunoreactive boutons. The similarity between the magno-and parvicellular axons in their targeting of postsynaptic elements, including the GABAergic neurons, suggests that the structural basis of the physiological differences between 4C alpha and 4C beta neurons should be sought in other aspects of the circuitry of layer 4C, such as local cortical circuits, or in the far greater horizontal extent of the thalamocortical and GABAergic axons in layer 4C alpha compared to those in the beta subdivision.     

Quantitative analysis of the choline acetyltransferase-immunoreactive axonal network in the cat primary visual cortex: I. Adult cats

The laminar distribution of cholinergic axons was analyzed quantitatively in the visual cortex of adult cats by using immunocytochemistry with a monoclonal antibody against choline acetyltransferase (ChAT). ChAT(+) fibers and varicosities were counted at different locations within area 17 and the distribution patterns in various animals were compared. Choline acetyltransferase-immunoreactivity was localized in fine, varicose fibers, which were present in all layers of the visual cortex. The density of labeled fibers was highest in layer I, which contained 14% of all fibers and 19.5% of all varicosities, and decreased toward deeper layers. The number of varicosities decreased more markedly toward deeper layers than the frequency of fibers. These distribution patterns were very consistent, showing only slight intra- and interindividual variability.     

Effects of stimulation of the dorsocaudal claustrum on activities of striate cortex neurons in the cat

In the cat striate cortex, single electrical shocks applied to the dorsocausal claustrum (CLdc) elicited bimodal excitatory responses with about 12 and 26 ms latencies. About one-fourth of the cortical cells observed had CLdc-induced inhibitions with onset latencies longer than the excitations. On cortical field responses to geniculate stimulation, CLdc conditioning shocks exerted early facilitatory and late inhibitory effects which were shown to be not transmitted through the mesencephalic reticular formation.     

Postnatal changes in the distribution of acetylcholinesterase in kitten striate cortex

We have traced the postnatal development of axons and cells in kitten striate cortex that contain acetylcholinesterase (AChE) by using a modification of Koelle's histochemical method. The maturation of AChE-positive axons was not found to be fully complete until at least 3 months of age, and was characterized by several distinct developmental trends. AChE-positive fibers in layers IVc-VI proliferate rapidly after birth until, by 4 weeks postnatal, they appear to exceed the adult density. They remain at this level as late as 8 weeks and then decrease to the adult density by 13 weeks. In contrast, the AChE-positive fibers in layer I do not show a substantial increase in density until 6 weeks of age and the adult level is not achieved before 3 months postnatal. Finally, the density of AChE-positive fibers in layers II and III appears to increase gradually from birth until the mature pattern is reached at about 6 weeks. AChE could also be localized histochemically to cell bodies whose position and appearance depended on postnatal age. Stained cells first appeared in the white matter subjacent to layer VI shortly after birth. By 2 weeks of age, most cells in layer VI were also AChE positive. The staining of these cells gradually disappears over the next 2 months until, at 3 months of age, there are no AChE-positive cells in cat striate cortex. However, a subpopulation of stained neurons appears in layer V by 1 year of age that persists throughout adulthood. The possible contributions of acetylcholine and AChE to the postnatal development of kitten striate cortex are discussed.     

Spiny neurons lacking choline acetyltransferase immunoreactivity are major targets of cholinergic and catecholaminergic terminals in rat striatum

The ultrastructural substrate for functional interactions between intrinsic cholinergic neurons and catecholaminergic afferents to the caudate-putamen nucleus and nucleus accumbens septi (NAS) was investigated immunocytochemically. Single sections of glutaraldehyde-fixed rat brain were processed 1) for the immunoperoxidase labeling of a rat monoclonal antibody against the acetylcholine-synthesizing enzyme choline acetyltransferase (CAT) and 2) for the immunoautoradiographic localization of a rabbit polyclonal antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). The ultrastructural morphology and cellular associations did not significantly differ in the caudate-putamen versus NAS. Immunoperoxidase reaction for CAT versus NAS. Immunoperoxidase reaction for CAT was seen in perikarya, dendrites, and terminals, whereas immunoautoradiography for TH was in terminals. The perikarya and dendrites immunolabeled for CAT were large, sparsely spiny, and postsynaptic mainly to unlabeled axon terminals. Only 2-3% of the CAT-labeled terminals (n = 136) and less than 1% of the TH-labeled terminals (n = 86) were apposed to, or formed synapses with, perikarya or dendrites immunoreactive for CAT. Most unlabeled and all labeled terminals formed symmetric synapses. In the same sample, 18% of the CAT and 16% of the TH-labeled terminals were directly apposed to each other. Unlabeled dendritic shafts received the major (40% for CAT versus 23% for TH) synaptic input from cholinergic terminals, while unlabeled spines received the major (47% for TH versus 23% for CAT) synaptic input from catecholaminergic terminals. Neither the unlabeled dendrites or spines received detectable convergent input from CAT and TH-labeled terminals. Thirteen percent of the CAT-labeled and 14% of TH-labeled terminals were in apposition to unlabeled terminals forming asymmetric, presumably excitatory, synapses with unlabeled dendritic spines. We conclude that in both the caudate-putamen and NAS cholinergic and catecholaminergic terminals 1) form symmetric, most likely inhibitory, synapses primarily with non-cholinergic neurons, 2) differentially synapse on shafts or spines of separate dendrites, and 3) have axonal appositions suggesting the possibility of presynaptic physiological interactions. These results support the hypothesis that the cholinergic-dopaminergic balance in striatal function may be mediated through inhibition of separate sets of spiny projection neurons with opposing excitatory and inhibitory functions.     

Involvement of GABAergic and cholinergic medial septal neurons in hippocampal theta rhythm

Hippocampal theta rhythm (HPCtheta) may be important for various phenomena, including attention and acquisition of sensory information. Two types of HPCtheta (types I and II) exist based on pharmacological, behavioral, and electrophysiological characteristics. Both types occur during locomotion, whereas only type II (atropine-sensitive) is present under urethane anesthesia. The circuit of HPCtheta synchronization includes the medial septum-diagonal band of Broca (MSDB), with cholinergic and gamma-aminobutyric acid (GABA)ergic neurons comprising the two main projections from MSDB to HPC. The primary aim of the present study was to assess the effects of GABAergic MSDB lesions on urethane- and locomotion-related HPCtheta, and compare these effects to those of cholinergic MSDB lesions. Saline, kainic acid (KA), or 192 IgG-saporin (SAP) was injected into MSDB before recording. KA preferentially destroys GABAergic MSDB neurons, whereas SAP selectively eliminates cholinergic MSDB neurons. A fixed recording electrode was placed in the dentate mid-molecular layer, and stimulating electrodes were placed in the posterior hypothalamus (PH), and medial perforant path (PP). Under urethane anesthesia, HPCtheta was induced by tail pinch, PH stimulation, and systemic physostigmine; none of the rats with KA or SAP showed HPCtheta in any of these conditions. During locomotion, HPCtheta was attenuated, but not eliminated, in rats with KA or SAP lesions. Intraseptal KA in combination with either intraseptal SAP or PP lesions reduced locomotion-related HPCtheta beyond that observed with each lesion alone, virtually eliminating HPCtheta. In contrast, intraseptal SAP combined with PP lesions did not reduce HPCtheta beyond the effect of each lesion alone. We conclude that both GABAergic and cholinergic MSDB neurons are necessary for HPCtheta under urethane, and that each of these septohippocampal projections contributes to HPCtheta during locomotion.     

Ultrastructure and synaptic targets of tectothalamic terminals in the cat lateral posterior nucleus

The recent appreciation of the fact that the pulvinar and lateral posterior (LP) nuclei receive two distinct types of cortical input has sparked renewed interest in this region of the thalamus. A key question is whether the primary or "driving" inputs to the pulvinar/LP complex originate in cortical or subcortical areas. To begin to address this issue, we examined the synaptic targets of tectothalamic terminals within the LP nucleus. Tectothalamic terminals were labeled using the anterograde transport of biotinylated dextran amine (BDA) or Phaselous leucoagglutinin placed in the superior colliculus or using immunocytochemical staining for substance P, a neurotransmitter found to be used by the tectothalamic pathway (Hutsler and Chalupa [ 1991] J. Comp. Neurol. 312:379-390). Our results suggest that most tectothalamic terminals are large and occupy a proximal position on the dendritic arbor of LP relay cells. In the medial LP, tectothalamic terminals labeled by the transport of neuronal tracers or substance P immunocytochemistry can form tubular clusters that surround the proximal dendrites of relay cells. In a rostral and lateral subdivision of the lateral LP nucleus (LPl-2), tectothalamic terminals form more typical glomerular arrangements. When compared with existing physiological data, these results suggest that a unique integration of tectal and cortical inputs may contribute to the response properties of LP neurons.     

Nerve growth factor receptor and choline acetyltransferase colocalization in neurons within the rat forebrain: response to fimbria-fornix transection

Although it is well known that magnocellular cholinergic basal forebrain neurons are trophically responsive to nerve growth factor (NGF) and contain NGF receptors (NGFr), the exact distribution of forebrain NGFr-immunoreactive neurons and the degree to which cholinergic neurons are colocalized with them have remained in question. In this study we employed a very sensitive double-labelling method and examined in the same tissue section the distribution and cellular features of NGFr-positive and choline acetyltransferase (ChAT)-immunolabelled neurons within the rat basal forebrain. Throughout this region the majority of magnocellular basal forebrain neurons were immunoreactive for both NGFr and ChAT. However, a small percentage of neurons in the ventral portion of the vertical limb of the diagonal band of Broca were immunoreactive only for NGFr, whereas a larger population of magnocellular neurons in the substantia innominata exhibited only ChAT immunoreactivity. No NGFr-immunoreactive cells were found associated with ChAT-positive neurons in the striatum, neocortex, or hippocampus, and no single-labelled NGFr-immunoreactive neurons were found outside the basal forebrain area, except for a large number of positive-labelled cells along the ventricular walls of the third ventricle. In addition to its function in maintaining the normal integrity of the basal forebrain and cholinergic, peripheral sympathetic, and neural-crest-derived sensory neurons, NGF may also have a role in the growth of these neurons after damage to the nervous system. To examine this postulate the hippocampus was denervated of its septal input and examined 8 weeks later. Two populations of neurons were found to have undergone collateral sprouting--namely, the midline magnocellular cholinergic neurons of the dorsal hippocampus and the sympathetic noradrenergic neurons of the superior cervical ganglion. Both of these neuronal populations also stained strongly for NGFr. In contrast, the small intrinsic cholinergic neurons of the hippocampus exhibited neither sprouting response nor staining for NGFr. In view of these results, we suggest that the differing sprouting responses demonstrated by these three neuronal populations may be due to their responsiveness to NGF, as indicated by the presence or absence of NGF receptors.     

Development of cholinergic and GABAergic neurons in the rat medial septum: Different onset of choline acetyltransferase and glutamate decarboxylase mRNA expression

In the present study, we have investigated the developmental expression of the transmitter-synthesizing enzymes choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in rat medial septal neurons by using in situ hybridization histochemistry. In addition, we have employed immunostaining for ChAT and the calcium-binding protein parvalbumin, known to be contained in septohippocampal GABAergic neurons. A large number of GAD67 mRNA-expressing neurons were already observed in the septal complex on embryonic day (E) 17, the earliest time point studied. During later developmental stages, there was mainly an increase in the intensity of labeling. Neurons expressing ChAT mRNA were first recognized at E 20, and their number slowly increased during postnatal development of the septal region. The adult pattern of ChAT mRNA-expressing neurons was observed around postnatal day (P) 16. By using a monoclonal ChAT antibody, the first immunoreactive cells were not seen before P 8. Similarly, the first weakly parvalbumin-immunoreactive neurons were seen in the septal complex by the end of the 1st postnatal week. These results indicate that in situ hybridization histochemistry may be an adequate method to monitor the different development of transmitter biosynthesis in cholinergic and GABAergic septal neurons. Moreover, the late onset of ChAT mRNA expression would be compatible with a role of target-derived factors for the differentiation of the cholinergic phenotype. © 1996 John Wiley-Liss, Inc.     

Organization and synaptic connections of cholinergic fibers in the cat superior colliculus

The cat superior colliculus (SC) receives a dense cholinergic input from three brainstem nuclei, the pedunculopontine tegmental nucleus, the lateral dorsal tegmental nucleus, and the parabigeminal nucleus (PBG). The tegmental inputs project densely to the intermediate gray layer (IGL) and sparsely to the superficial layers. The PBG input probably projects only to the superficial layers. In the present study, the morphology of choline acetyltransferase (ChAT)-immunoreactive axons and synaptic endings in the superficial and deep layers of the SC was examined by light and electron microscopy to determine whether these cholinergic afferents form different types of synapses in the superifical and deep layers. Two types of fibers were found within the zonal (ZL) and upper superficial gray layers (SGL): small diameter fibers with few varicosities and larger diameter fibers with numerous varicosities. Quantitative analysis demonstrated a bimodal distribution of axon diameters, with one peak at approximately 0.3–0.5 μm and the other at 0.9–1.0 μm. On the other hand, ChAT-immunoreactive fibers in the IGL were almost all small and formed discrete patches within the IGL. Two types of ChAT-immunoreactive synaptic profiles were observed within the ZL and upper SGL using the electron microscope. The first type consisted of small terminals containing predominantly round synaptic vesicles and forming asymmetric synaptic contacts, mostly on dendrites. The second type was comprised of varicose profiles that also contained round synaptic vesicles. Their synaptic contacts were always symmetric in profile. ChAT-immunoreactive terminals in the IGL patches contained round or pleomorphic synaptic vescles, and the postsynaptic densities varied from symmetric to asymmetric, including intermediate forms. However, no large varicose profiles were observed. This study suggests that cholinergic fibers include at least two differnt synaptic morphologies: small terminals with asymmetric thickenings and large varicose profiles with symmetric terminals. The large varicose profile in the superficial layers is absent in the IGL. This result suggests that the cholinergic inputs that innervate the superficial layers and the patches in the IGL of the cat SC differ in their synaptic organization and possibly also in their physiological actions. © 1993 Wiley-Liss, Inc.     

Long-range GABAergic projection neurons in the cat neocortex

Neocortical gamma-aminobutyric acid (GABA)ergic neurons have been previously described as largely involved in local intracortical circuitry. However, our recent findings in the murine model described select neocortical GABAergic neurons that project to both neighboring and more distant neocortical regions. Here, we investigated whether such GABAergic projection neurons are also found in the cat neocortex. Wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the visual, auditory, or somatosensory cortex, in order to label efferent cortical neurons retrogradely and to label axons and terminals orthogradely. Staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), an enzyme involved in nitric oxide synthesis, was employed, and co-localization with WGA-HRP was determined by means of both polarizing and brightfield microscopy. We concluded that neurons double-labeled with WGA-HRP and NADPH-d in a distant region from the WGA-HRP-injection site are GABAergic neurons with long-range projection axons. All double-labeled neurons were found in cortical layers VIa and VIb and in the white matter. Neurons with intense NADPH-d reactivity (type I) were determined to be neuronal nitric oxide synthase (nNOS) positive in all cases. However, weakly NADPH-d-reactive neurons (type II) lacked nNOS immunoreactivity. Moreover, nNOS often co-localized with GABA, neuropeptide-Y, and somatostatin in the cat neocortex. In summary, the GABAergic neurons described here projected in a manner similar to that previously described for neocortical principal neurons, although some unique GABAergic long-range projections were also demonstrated.