Descending projections from the basal forebrain to the orexin neurons in mice

The orexin (hypocretin) neurons play an essential role in promoting arousal, and loss of the orexin neurons results in narcolepsy, a condition characterized by chronic sleepiness and cataplexy. The orexin neurons excite wake‐promoting neurons in the basal forebrain (BF), and a reciprocal projection from the BF back to the orexin neurons may help promote arousal and motivation. The BF contains at least three different cell types (cholinergic, glutamatergic, and γ‐aminobutyric acid (GABA)ergic neurons) across its different regions (medial septum, diagonal band, magnocellular preoptic area, and substantia innominata). Given the neurochemical and anatomical heterogeneity of the BF, we mapped the pattern of BF projections to the orexin neurons across multiple BF regions and neuronal types. We performed conditional anterograde tracing using mice that express Cre recombinase only in neurons producing acetylcholine, glutamate, or GABA. We found that the orexin neurons are heavily apposed by axon terminals of glutamatergic and GABAergic neurons of the substantia innominata (SI) and magnocellular preoptic area, but there was no innervation by the cholinergic neurons. Channelrhodopsin‐assisted circuit mapping (CRACM) demonstrated that glutamatergic SI neurons frequently form functional synapses with the orexin neurons, but, surprisingly, functional synapses from SI GABAergic neurons were rare. Considering their strong reciprocal connections, BF and orexin neurons likely work in concert to promote arousal, motivation, and other behaviors. J. Comp. Neurol. 525:1668–1684, 2017. © 2016 Wiley Periodicals, Inc.     

Activation of orexin/hypocretin projections to basal forebrain and paraventricular thalamus by acute nicotine

Orexin/hypocretin neurons of the lateral hypothalamus/perifornical area project to a diverse array of brain regions and are responsive to a variety of psychostimulant drugs. It has been shown that orexin neurons are activated by systemic nicotine administration suggesting a possible orexinergic contribution to the effects of this drug on arousal and cognitive function. The basal forebrain and paraventricular nucleus of the dorsal thalamus (PVT) both receive orexin inputs and have been implicated in arousal, attention and psychostimulant drug responses. However, it is unknown whether orexin inputs to these areas are activated by psychostimulant drugs such as nicotine. Here, we infused the retrograde tract tracer cholera toxin B subunit (CTb) into either the basal forebrain or PVT of adult male rats. Seven to 10 days later, animals received an acute systemic administration of (-) nicotine hydrogen tartrate or vehicle and were euthanized 2h later. Triple-label immunohistochemistry/immunofluorescence was used to detect Fos expression in retrogradely-labeled orexin neurons. Nicotine increased Fos expression in orexin neurons projecting to both basal forebrain and PVT. The relative activation in lateral and medial banks of retrogradely-labeled orexin neurons was similar following basal forebrain CTb deposits, but was more pronounced in the medial bank following PVT deposits of CTb. Our findings suggest that orexin inputs to the basal forebrain and PVT may contribute to nicotine effects on arousal and cognition and provide further support for the existence of functional heterogeneity across the medial-lateral distribution of orexin neurons.     

Activation of orexin/hypocretin projections to basal forebrain and paraventricular thalamus by acute nicotine

Orexin/hypocretin neurons of the lateral hypothalamus/perifornical area project to a diverse array of brain regions and are responsive to a variety of psychostimulant drugs. It has been shown that orexin neurons are activated by systemic nicotine adminstration suggesting a possible orexinergic contribution to the effects of this drug on arousal and cognitive function. The basal forebrain and paraventricular nucleus of the dorsal thalamus (PVT) both receive orexin inputs and have been implicated in arousal, attention and psychostimulant drug responses. However, it is unknown whether orexin inputs to these areas are activated by psychostimulant drugs such as nicotine. Here, we infused the retrograde tract tracer cholera toxin B subunit (CTb) into either the basal forebrain or PVT of adult male rats. Seven to 10 days later, animals received an acute systemic administration of (−) nicotine hydrogen tartrate or vehicle and were euthanized 2 h later. Triple-label immunohistochemistry/immunofluorescence was used to detect Fos expression in retrogradely-labeled orexin neurons. Nicotine increased Fos expression in orexin neurons projecting to both basal forebrain and PVT. The relative activation in lateral and medial banks of retrogradely-labeled orexin neurons was similar following basal forebrain CTb deposits, but was more pronounced in the medial bank following PVT deposits of CTb. Our findings suggest that orexin inputs to the basal forebrain and PVT may contribute to nicotine effects on arousal and cognition and provide further support for the existence of functional heterogeneity across the medial-lateral distribution of orexin neurons.     

Effects of hypocretin (orexin) neuronal loss on sleep and extracellular adenosine levels in the rat basal forebrain

Neurons containing the neuropeptide hypocretin (HCRT, orexin) are localized only in the lateral hypothalamus, from where they innervate multiple regions implicated in arousal, including the basal forebrain. HCRT activation of downstream arousal neurons is likely to stimulate release of endogenous factors. One such factor is adenosine, which in the basal forebrain increases in level with wakefulness and decreases with sleep, and is hypothesized to regulate the waxing and waning of sleep drive. Does loss of HCRT neurons affect adenosine levels in the basal forebrain? Is the increased sleep that accompanies HCRT loss a consequence of higher adenosine levels in the basal forebrain? In the present study, we investigated these questions by lesioning the HCRT neurons with HCRT‐2–saporin (HCRT‐2–SAP) and measuring sleep and extracellular levels of adenosine in the basal forebrain. In separate groups of rats, the neurotoxin HCRT‐2–SAP or saline was administered locally to the lateral hypothalamus, and 80 days later adenosine and sleep were assessed. Rats given the neurotoxin had a 94% loss of HCRT neurons. These rats woke less at night, and had more rapid eye movement sleep, which is consistent with HCRT hypofunction. These rats also had more sleep after brief periods of sleep deprivation. However, in the lesioned rats, adenosine levels did not increase with 6 h of sleep deprivation, whereas an increase in adenosine levels occurred in rats without lesion of the HCRT neurons. These findings indicate that adenosine levels do not increase with wakefulness in rats with a HCRT lesion, and that the increased sleep in these rats occurs independently of adenosine levels in the basal forebrain.     

Inhibition of rostral basal forebrain neurons promotes wakefulness and induces FOS in orexin neurons

The present study examined whether the activities of the rostral basal forebrain neurons alter the activities of the orexin (also known as hypocretin) neurons in the tuberal part of the hypothalamus in rats. We performed microdialysis perfusion of the ventromedial portion of the rostral basal forebrain with the GABAA receptor agonist muscimol to inhibit focally the neuronal activities in the rostral basal forebrain. Then, we monitored sleep/wake behaviour and investigated the pattern of activities of orexin neurons by examining the expression of FOS as an indicator of cellular activation. Bilateral perfusion with muscimol (5, 15, and 50 micro m) produced a dose-dependent decrease in the amount of sleep. This perfusion with muscimol at 50 micro m produced FOS-like immunoreactivity in 37% of the orexin neurons located in the tuberal part of the hypothalamus, whereas the FOS-like immunoreactivity was sparse in orexin neurons of the sleeping control rats (P = 0.001 by Mann-Whitney U-test). Unilateral perfusion with muscimol (50 micro m) also suppressed sleep. In this case, FOS-like immunoreactivity was seen in 40% of the orexin neurons on the side ipsilateral to the perfusion site but only in 10% of orexin neurons on the contralateral side (P = 0.018 by Wilcoxon signed rank test). These functional data suggested that a sleep-generating element in the ventromedial part of the rostral basal forebrain provides an inhibitory influence on the activities of the orexin neurons in the tuberal part of the hypothalamus.     

Topographic projections to the visual cortex from the basal forebrain in the rat

Recent studies have shown that cholinergic neurons within the basal forebrain give rise to a projection that terminates throughout the neocortex. The purpose of the present study is to determine the topographic organization of the basal forebrain projection to a single cortical region: the visual cortex. Injections of wheat germ agglutinin-horseradish peroxidase were placed within the infragranular layers of areas 17, 18a or 18b in Long-Evans hooded rats. Following injections placed within area 18a, retrogradely labeled neurons were located primarily within the caudal components of the basal forebrain including the basal nucleus of Meynert. Injections placed within area 18b, on the other hand, resulted in retrograde labeling of numerous neurons within rostral basal forebrain nuclei, including the horizontal limb of the diagonal band of Broca, whereas only a few labeled cells were located within the basal nucleus. Two patterns of labeled cells were evident following injections placed within area 17 and they resembled the results from injections within the adjacent extrastriate area; i.e. either area 18a or area 18b. Thus, injections restricted to the lateral portions of area 17 resulted in retrograde labeling of neurons located within the caudal levels of the basal forebrain, whereas injections within the medial portions of area 17 were followed by retrograde labeling of neurons within the rostral levels of the basal forebrain. Based on these results we suggest that the cholinergic corticopetal projection terminates within the visual cortex in a medial to lateral pattern.     

Neurogenesis of basal forebrain cholinergic neurons in rat

The basal forebrain cholinergic system embodies a heterogeneous group of neurons distributed in the basal telencephalon that project topographically to the cortical mantle. We sought to examine the generation of these neurons to determine whether basal forebrain neurons have unique patterns of neurogenesis or, if, in contrast, they are born along general neurogenic gradients. The techniques of tritiated thymidine autoradiography and choline acetyltransferase (ChAT) immunocytochemistry were combined to determine the birthdays and neurogenic gradients of cholinergic cells in this region of rat brain. Cholinergic neurogenesis throughout the basal forebrain ranged from embryonic days 12 to 17 (E12-17). Neurogenesis in the nucleus basalis magnocellularis occurred over E12-16, with a peak day of generation on E13. The horizontal limb nucleus of the diagonal band which is located rostral to the nucleus basalis was generated over E12-17, with the majority of cells arising on E14-15. The rostral-most nuclei of the basal forebrain cholinergic system, the vertical limb of the diagonal band and the medial septum, were generated between E13-17, with peak days of neurogenesis on E15 and E15-16, respectively. These results were evaluated quantitatively and demonstrated that the basal forebrain cholinergic neurons were generated along the general caudal-to-rostral gradient previously described for all neurons in this brain region. The results of this study, in combination with those of similar investigations, emphasize that position-dependent epigenetic factors appear to be more potent determinants of the time of neuronal origin than factors which influence a cell's transmitter phenotype.     

Cholinergically mediated reduction of locomotor activity from the basal forebrain of the rat

Carbachol when injected into the basal forebrain alters spontaneous motor behavior and usually decreases locomotion. However, the extent of the brain area producing this effect has not yet been determined. The goal of the present study was to use quantitative mapping of injection sites to further localize the effect of carbachol on spontaneous locomotion of rats. The distance travelled by an animal and the time spent moving were simultaneously measured before and after injection of carbachol or saline into 96 sites in the basal forebrain. Each site was injected with 1.0 microgram (5.47 nmol) of carbachol, a dose close to ED50, in a volume of 0.2 microliter. A decrease in spontaneous locomotion was obtained as a result of injections of carbachol into the preoptic and anterior hypothalamic areas, particularly into the medial preoptic nucleus and the latero-anterior hypothalamic nucleus. The area from which a consistent decrease in spontaneous locomotion was obtained was surrounded by an area producing an increase in locomotion with a narrow zone of overlap. This decrease in locomotion was dose dependent and reversed by atropine. The results indicate that both the decreasing and increasing effects of carbachol on locomotion are anatomically specific and that the decreasing effects can be elicited from a limited forebrain area. It is suggested that muscarinic cholinergic mechanisms in the basal forebrain may be involved in the pathogenesis of neural dysfunction associated with locomotor activity in man.     

Evidence for basal forebrain thirst osmoreceptors in rat

Albino rats received bilateral intracranial injections of 0.33 or 0.34osM saline or sucrose solutions through injectors that terminated either in the lateral ventricles, the bed nuclei of the stria terminalis or in the lateral preoptic area. Both solutions elicited drinking when injected into the bed nuclei of the stria terminalis or lateral preoptic area but not when injected into the lateral ventricles. Only 0.48osM saline was effective intraventricularly. These findings suggest that there are thirst osmoreceptors in the basal forebrain that can be activated by hypertonic solutions that are within the physiological range. They do not support the view that mammals are apprised of cellular dehydration by ventricular Na receptors.     

Organization of hypocretin/orexin efferents to locus coeruleus and basal forebrain arousal-related structures

Hypocretin/orexin neurons give rise to an extensive projection system, portions of which innervate multiple regions associated with the regulation of behavioral state. These regions include the locus coeruleus, medial septal area, medial preoptic area, and substantia innominata. Evidence indicates that hypocretin modulates behavioral state via actions within each of these terminal fields. To understand better the circuitry underlying hypocretin-dependent modulation of behavioral state, the present study characterized the degree to which there exists: 1) lateralization of hypocretin efferents to basal forebrain and brainstem arousal-related regions, 2) topographic organization of basal forebrain- and brainstem-projecting hypocretin neurons, and 3) collateralization of individual hypocretin neurons to these arousal-related terminal fields. These studies utilized combined immunohistochemical identification of hypocretin neurons with single or double retrograde tracing from the locus coeruleus, medial preoptic area, medial septal area, and substantia innominata. Results indicate that approximately 80% of hypocretin efferents to basal forebrain regions project ipsilaterally, whereas projections to the locus coeruleus are more bilateral (65%). There was a slight preference for basal forebrain-projecting hypocretin neurons to be distributed within the medial half of the hypocretin cell group. In contrast, hypocretin neurons projecting to the locus coeruleus were located primarily within the dorsal half of the hypocretin cell group. Finally, a large proportion of hypocretin neurons appear to project simultaneously to at least two of the examined terminal fields. These latter observations suggest coordinated actions of hypocretin across multiple arousal-related regions.     

Time of origin of cholinergic neurons in the rat basal forebrain

The timing of the final mitotic division of basal forebrain cholinergic neurons was studied by injecting [3H]thymidine into timed pregnant rats and processing the brains of their progeny as young adults for immunohistochemistry with a monoclonal antibody to choline acetyltransferase (ChAT) followed by autoradiography. ChAT-positive neurons located caudally in the basal forebrain were found to become postmitotic mostly on embryonic (E) days 12 and 13, whereas the peak final mitosis of more rostrally located ChAT-positive neurons occurred increasingly later, with the most rostral ChAT-immunoreactive neurons leaving their final mitotic cycles on E15 and E16. In all basal forebrain regions, cholinergic neurogenesis was complete by E17. These results indicate that the cholinergic neurons in the basal forebrain become postmitotic in a caudal-to-rostral gradient over about 5 days. The continuity of the gradient suggests that these cholinergic neurons may derive from the same germinal source.     

Activity of basal forebrain neurons in the rat during motivated behaviors

The activity of single neurons in the basal forebrain was recorded in the freely-moving rat with moveable fine-wire electrodes. Neural activity was observed while the water-deprived male rat was exposed to three different types of motivating stimuli that elicit locomotion in a running wheel: an estrous female rat; a drinking tube containing water; and grasping and lifting by the experimenter. The neural activity was also observed when the subject was presented with standardized sensory tests and during single pulse stimulation of other brain structures. A majority of the 76 neurons recorded in the forebrain changed their firing rate during orienting and/or locomotion in general (23 neurons) or during behavior related to only one of the specific motivational contexts: the conspecific female (4 neurons); water (7 neurons); or grasp by the experimenter (8 neurons). Whereas the neurons related to orienting and/or locomotion in general were scattered through various brain structures, those neurons related to specific motivational contexts were concentrated in specific areas: the sexually dimorphic nucleus of the medial preoptic area (conspecific female); lateral septum (water); and lateral preoptic area (water and grasp). The present results, although based on relatively few neurons, are consonant with results of research using other techniques. This indicates that analyses at the level of the single neuron promise to be useful for understanding the role of the basal forebrain in motivational systems.     

Galanin-immunoreactive synaptic terminals on basal forebrain cholinergic neurons in the rat

Previous studies have indicated that galanin is one of the most abundant peptides in the basal forebrain and that it has a significant modulatory influence on cholinergic transmission. The aim of the present study was to use a light electron microscopic correlation technique to determine whether galanin-immunoreactive terminals form synaptic contacts with basal forebrain cholinergic cells of the rat. Sections from fixed-perfused brains were stained at the light and electron microscopic levels for galanin and choline acetyltransferase immunoreactivity in the same section by using a dual-colour immunohistochemical method. The results showed that galanin-immunoreactive axonal terminals are unevenly distributed in the medial septal nucleus, the diagonal band, and the nucleus basalis. Galanin-positive synapses were most prominent on choline acetyltransferase-positive neurons in the lateral parts of the nucleus of the diagonal band and in the posterior half of the nucleus basalis, which is where there was the greatest overlap between the distribution of galanin-immunoreactive terminals and choline acetyltransferase-positive neurons. The origins of these galanin-positive terminals are not known, but the results confirm that the basal forebrain galaninergic system has a synaptic influence on basal forebrain cholinergic neurons in the rat. J. Comp. Neurol. 383:82–93, 1997. © 1997 Wiley-Liss, Inc.     

The basal forebrain cholinergic system and object memory in the rat

Rats with near complete destruction of basal forebrain cholinergic neurons from intracerebroventricular injections of 192 IgG-saporin were trained on object discrimination problems and then retrained two weeks later to measure retention. Despite dramatic reductions of acetylcholinesterase-positive fibers in hippocampus and neocortex, these animals did not differ from controls on an analysis of savings scores. Thus, the basal forebrain cholinergic system may serve functions that support non-spatial memory but are not specifically mnemonic in nature.     

Effect of basal forebrain somatostatin and parvalbumin neurons in propofol and isoflurane anesthesia

AimsThe basal forebrain (BF) plays an essential role in wakefulness and cognition. Two subtypes of BF gamma‐aminobutyric acid (GABA) neurons, including somatostatin‐expressing (GABA^SOM) and parvalbumin‐positive (GABA^Parv) neurons, function differently in mediating the natural sleep–wake cycle. Since the loss of consciousness induced by general anesthesia and the natural sleep–wake cycle probably share similar mechanisms, it is important to clarify the accurate roles of these neurons in general anesthesia procedure.MethodsBased on two transgenic mouse lines expressing SOM‐IRES‐Cre and PV‐IRES‐Cre, we used a combination of genetic activation, inactivation, and chronic ablation approaches to further explore the behavioral and electroencephalography (EEG) roles of BF^SOM and BF^Parv neurons in general anesthesia. After a single intravenous injection of propofol and the induction and recovery times of isoflurane anesthesia, the anesthesia time was compared. The changes in cortical EEG under different conditions were also compared.ResultsActivation of BF GABA^SOM neurons facilitates both the propofol and isoflurane anesthesia, manifesting as a longer anesthesia duration time with propofol anesthesia and a fast induction time and longer recovery time with isoflurane anesthesia. Moreover, BF GABA^SOM‐activated mice displayed a greater suppression of cortical electrical activity during anesthesia, showing an increase in δ power bands or a simultaneous decrease in high‐frequency power bands. However, only a limited and nuanced effect on propofol and isoflurane anesthesia was observed with the manipulated BF GABA^Parv neurons.ConclusionsOur results suggested that BF GABA^SOM neurons play a critical role in propofol and isoflurane general anesthesia, while BF GABA^Parv neurons appeared to have little effect.     

Attenuation of the blood flow response to physostigmine in the rat cortex deafferented from the basal forebrain

Previous functional investigations in rats failed to demonstrate that the classical cholinesterase inhibitor, physostigmine, can compensate for cortical cholinergic deficit induced by deafferentation from the nucleus basalis magnocellularis (NBM). As these studies were carried out shortly after NBM lesion (1-2 weeks), we sought to determine whether compensatory effects of physostigmine would appear at a longer postlesion time (3-5 weeks). Cerebral blood flow was used as a quantitative measure of brain function. At 3-5 weeks after unilateral NBM lesion, interhemispheric comparisons in resting conditions showed that the cortical cholinergic deficit was still present and that blood flow was lower in cortical areas on the lesion side, similarly to what was observed after 1-2 weeks, while basal blood flow in intact hemispheres remained unchanged. In contrast, under physostigmine, blood flow became significantly lower in deafferented cortical areas at 3-5 weeks postlesion time, whereas there were no significant interhemispheric differences in the short term. Comparisons with saline-infused rats showed reduced blood flow responses to physostigmine in forebrain regions, e.g. in the parietal cortex from 83% to 25% at 1-2 and 3-5 weeks postlesion, respectively. These changes cannot be ascribed to a global loss of reactivity, since responses in brainstem regions (medulla, cerebellum) remained unchanged statistically. The results demonstrate a reduced responsiveness to physostigmine at the longer postlesion time, and support the existence of a cholinosensitive mechanism antagonizing NBM influence. This mechanism may limit the activating effects of cholinergic agonists in the forebrain after NBM deafferentation.     

Codistribution of GABA- with acetylcholine-synthesizing neurons in the basal forebrain of the rat

In recent years, GABAergic neurons have been identified in the basal forebrain where cholinergic cortically projecting neurons are located and known to be important in mechanisms of cortical activation. In the present study in the rat, the relationship of the GABA-synthesizing neurons to the acetylcholine-synthesizing neurons was examined by application of a sequential double staining immunohistochemical procedure involving the peroxidase-antiperoxidase technique for glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT). In these double and adjacent single immunostained series of sections, the GAD+ and ChAT+ cells were mapped, counted and measured with the aid of a computerized image analysis system. Through the entire basal forebrain, there was no evidence for colocalization of GAD and ChAT in the same neurons. Instead, a large population of GAD-immunoreactive neurons is codistributed with ChAT-immunoreactive neurons and outnumbers them by a factor of two: approximately 39,000 GAD+ cells to 18,000 ChAT+ cells. Although the GAD+ and ChAT+ neurons lie intermingled within fascicles of the major longitudinal and transverse forebrain fiber systems in subregions of the basal forebrain, the GAD+ cells are more highly concentrated within different sectors of the pathways and regions than the ChAT+ cells. Although GAD+ neurons resemble ChAT+ neurons in certain regions, both being bi- or multipolar and, on average, medium-sized cells, the GAD+ neurons are, in the majority (51%), small-sized cells ( < 15 μm in length) and as a population significantly smaller than the ChAT+ neurons. These results suggest that many GABAergic neurons may represent interneurons in the basal forebrain and potentially exert an inhibitory influence on adjacent cortically projecting cholinergic neurons. Medium- to large GAD+ cells, which resemble similar ChAT+ cells, are also present and represent the majority of the GAD+ cells in the nucleus of the diagonal band of Broca, magnocellular preoptic nucleus, and olfactory tubercle, but represent the minority in the anterior and posterior substantia innominata and globus pallidus. Given their prominent size, such GABAergic cells may also exert an inhibitory influence outside the basal forebrain as projection neurons and potentially in parallel with cholinergic neurons, to certain regions of the cerebral cortex. Accordingly, GABAergic cells may be considered as constituents of the magnocellular basal nucleus and potentially important elements within the ventral extrathalamic relay from the brainstem reticular formation to the cerebral cortex. © 1993 Wiley-Liss, Inc.     

Lesions of the basal forebrain in rat selectively impair the cortical vasodilation elicited from cerebellar fastigial nucleus

We sought to determine in rat, whether interruption of the major extrathalamic projections to the cerebral cortex originating in and projecting through the basal forebrain (BF), will impair the increase in regional cerebral blood flow (rCBF), but not metabolism, elicited in the cerebral cortex by electrical stimulation of the cerebellar fastigial nucleus (FN). Studies were conducted in anesthetized, paralyzed, ventilated rats, with blood gases controlled and AP maintained in the autoregulated range. Electrolytic lesions were placed unilaterally in the BF at the level of the lateral preoptic region lying in rostral portions of the medial forebrain bundle and resulted in a reduction of up to 47% of the choline acetyltransferase activity in the ipsilateral cerebral cortex. rCBF was measured in homogenates of 9 paired brain regions by the 14C-iodoantipyrine technique. In unlesioned rats, FN stimulation symmetrically and significantly (P less than 0.05) increased rCBF in all brain regions with the greatest increase (to 180%) in the frontal cortex. Two days following a unilateral BF lesion, FN stimulation failed to increase rCBF in the ipsilateral cerebral cortex distal to the BF lesion. In contrast, rCBF was increased to an almost comparable degree in the remainder of the brain. BF lesions alone resulted in a 18-23% reduction in cortical rCBF ipsilaterally (P less than 0.025). BF lesions did not alter the cerebrovascular vasodilation elicited by CO2 nor perturb autoregulation. The cortical vasodilation elicited by FN stimulation is mediated by intrinsic neuronal pathways and depends upon the integrity of neurons, possibly cholinergic, originating in, or passing through, the BF.     

Neurons in the basal forebrain complex of the rat: a Golgi study

Several types of neurons are coexistent in the basal forebrain nuclear complex of the rat. In the medial septum and vertical limb of the diagonal band 3 classes of neurons occur which are characterized by varicose dendrites. In the horizontal limb of the diagonal band neurons with smooth dendrites and those with varicose dendrites are intermingled. We found 3 classes of neurons in the nucleus preopticus magnocellularis. A giant type with smooth and varicose dendrites occurs in this nucleus, but also in the substantia innominata. In the substantia innominata-nucleus basalis complex 4 classes of neurons with varicose dendrites and 2 classes with spiny dendrites have been observed. Our findings suggest that the nucleus of the vertical limb of the diagonal band forms a unit with the nucleus septi medialis, but is separated from the nucleus of the horizontal limb of the diagonal band by different neuronal composition. The nucleus preopticus magnocellularis is a separate nuclear structure characterized by a content of neurons different from those in the horizontal limb of the diagonal band and in most components also from the substantia innominata-nucleus basalis complex. There is some evidence that the cholinergic neurons have to be searched among those with varicose dendrites.