Cholinergic neurons expressing substance P receptor (NK1) in the basal forebrain of the rat: a double immunocytochemical study

Cholinergic neurons expressing substance P receptor (SPR, NK₁) were examined in the rat brain using double immunofluorescence. The distribution of SPR-like immunoreactive (SPR-LI) neurons completely overlapped with that of choline acetyltransferase (ChAT)-LI neurons in the medial septal nucleus, the nucleus of diagonal band of Broca, the magnocellular preoptic nucleus, the substantia innominata of basal forebrain, the caudate-putamen, and the ventral pallidum of the basal ganglia. In the mesopontine tegmentum and the cranial motor nuclei of the brainstem, the distribution of SPR-LI and ChAT-LI neurons was partially overlapping. Neurons showing both SPR-like and ChAT-like immunoreactivities, however, were predominantly found above basal forebrain regions and 82–90% of these ChAT-LI neurons displayed SPR-like immunoreactivity, in addition to the confirmatory observation that 100% of the ChAT-LI neurons exhibit SPR-like immunoreactivity in the basal ganglia. In contrast, neurons double-labeled for SPR-like and ChAT-like immunoreactivities were hardly detected in aforementioned regions of the brainstem. The present study has provided morphological evidence for direct physiological modulation of cholinergic neurons by tachykinins through substance P receptor in the basal forebrain of the rat.     

Retinal dopaminergic neurons (A17) expressing neuromedin K receptor (NK(3)): a double immunocytochemical study in the rat

By using a double immunofluorescence method we examined the distribution of dopaminergic neurons (A17) expressing neuromedin K receptor (NKR, NK(3)) in the rat retina. The distribution of NKR-like immunoreactive (-LI) neurons partially overlapped that of tyrosine hydroxylase (TH)-LI neurons in the inner retina of section and flat-mount preparation. Neurons showing both TH- and NKR-like immunoreactivities were found in the retina (A17): 100% of these TH-LI neurons displayed NKR-like immunoreactivity, and they constituted about 3.5% of total NKR-LI neurons. The majority of double-labeled neurons with TH- and NKR-like immunoreactivities were distributed in the proximal inner nuclear layer and the upper part of inner plexiform layer of the retina, and characterized with appearance of amacrine cells. The present study has provided morphological evidence for direct physiological modulation of dopaminergic neurons by tachykinins through NKR in the rat retina (A17).     

Noradrenergic neurons expressing substance P receptor (NK1) in the locus coeruleus complex: a double immunofluorescence study in the rat

By using a double immunofluorescence method we examined the distribution of noradrenergic neurons expressing substance P receptor (NK1) or neuromedin K receptor (NK3) in the rat brainstem. The distribution of SPR-like immunoreactive (-LI) neurons completely overlapped that of tyrosine hydroxylase (TH)-LI neurons in the locus coeruleus (A6), ventrolateral and lateral reticular formation of pons (A5 and A7). Partially overlapping distribution of SPR- and TH-LI neurons were found in certain regions of the medulla oblongata (A1-A4). Neurons showing both SPR- and TH-like immunoreactivities, however, were only found in the locus coeruleus complex (A5-A7): 100% of these TH-LI neurons displayed SPR-like immunoreactivity. Neurons showing both NKR- and TH-like immunoreactivities were not detected in the aforementioned areas of brainstem. The present study has provided morphological evidence for direct physiological modulation of noradrenergic neurons by tachykinins through SPR in locus coeruleus complex (A5-A7).     

Substance P-Containing terminals in synaptic contact with cholinergic neurons in the neostriatum and basal forebrain: a double immunocytochemical study in the rat

Antibodies against substance P and choline acetyltransferase (ChAT) have been used in a sequential double-immunocytochemical ultrastructural study of the rat forebrain. The peroxidase-anti-peroxidase procedure was used for both antigens, however, two different substrates for the peroxidase reactions were used. The substance P-immunoreactive sites were first localized using 3,3'-diaminobenzidine as the substrate, then the ChAT-immunoreactive sites were localized using benzidine dihydrochloride. The reaction product formed by the two substrates was distinguishable in both the light and electron microscopes. Using this procedure, the cell bodies and proximal dendrites of identified cholinergic neurons in the neostriatum were found to receive symmetrical synaptic input from substance P-immunoreactive boutons. A similar pattern of substance P-immunoreactive synaptic input was observed onto magnocellular basal forebrain cholinergic neurons in the ventral pallidum and ventromedial globus pallidus. In both the striatum and basal forebrain substance P-immunoreactive boutons were also seen in contact with structures that did not display ChAT immunoreactivity.     

Mesencephalic dopaminergic neurons expressing neuromedin K receptor (NK3): a double immunocytochemical study in the rat

By using a double immunocytochemical method we examined the distribution of dopaminergic neurons expressing neuromedin K receptor (NKR; NK₃) in the rat brain. The distribution of NKR-like immunoreactive (-LI) neurons completely overlapped that of tyrosine hydroxylase (TH)-LI neurons in the retrorubral field (A8), substantia nigra (A9), ventral tegmental area and nucleus raphe linealis (A10). Completely or partially overlapping distributions of NKR- and TH-LI neurons were found in certain regions of the hypothalamus (A11–A15) and olfactory bulb (A16). Neurons showing both NKR- and TH-like immunoreactivities, however, were only found in A8–A10: All of the NKR-LI neurons displayed TH-like immunoreactivity, and about 71–86% of the TH-LI neurons expressed NKR-like immunoreactivity. The present results provided morphological evidence for physiological modulation of dopaminergic neurons by tachykinins through NKR in A8–A10.     

Marginal topography of neurons expressing the substance P receptor in the rat suprachiasmatic nucleus

Neurons expressing the substance P (SP) receptor (NK1 receptor) in the suprachiasmatic nucleus of the hypothalamus (SCN) have been topographically identified using radioactive in situ hybridization histochemistry. In the anterior hypothalamic area, clustered labeled neurons of small size and exhibiting low levels of gene expression are observed exclusively at the dorsolateral margin of the SCN, straddling cytoarchitectural boundaries of the nucleus. The marginal topography of neurons putative target of a SP-containing retinal input to the ventral SCN indicates that their dendrites bearing the receptor extend towards the retinorecipient part of the nucleus, where they can be modulated by overlapping inputs from the intergeniculate leaflet and the raphé. Eventual interactions between glutamatergic and putative tachykininergic retinal pathways for a coherent photic control of circadian rhythms may therefore occur mainly via intrinsic neuronal connections between their distinct target populations. In addition, since glutamate and SP induce electrophysiological responses in ventrolateral neurons with no interactive effect, neurons integrating both chemical signals, subsequently to their modulation by several influences, may be not located within the ventrolateral SCN. Alternatively but not exclusively, marginal neurons could be a target of SP-containing neurons within the SCN or nearby the nucleus, or from ascending projections from the raphé where serotonin and SP colocalize. The marginal topography of neurons expressing the SP receptor supports the view of the involvement of neurons located in the vicinity of the nucleus in the regulation of circadian rhythms.     

Cholinergic neurons in the basal forebrain of aged female mice

Aging is associated with at least down-regulation of several cellular functions and diminished responsiveness to internal and external signals, and possibly with direct cell death. Consequently, pharmacological manipulations may be less effective in aged than in young organisms. In the present study, we investigated whether the basal forebrain cholinergic neurons and the estrogen receptor alpha (ERalpha) which they contain respond to changes in estrogen availability in aged female mice. The mice were sham-operated, ovariectomized, or ovariectomized and treated with 17beta-estradiol at the age of 18 months. Three months later, the mice were perfused and brain sections were double immunostained for choline acetyltransferase (ChAT) and ERalpha. Cell counting with a stereological method revealed that changes in the estrogen level have no effect on the total number of ChAT-immunoreactive (ir) neurons in the basal forebrain. However, the percentage of ChAT-ir neurons containing ERalpha-ir was higher in the ovariectomized mice than in the sham-operated or estrogen-treated mice. This was specific for the medial septum and vertical diagonal band of Broca. The findings indicate that even at old age the ERalphas in cholinergic cells are able to respond to changes in estrogen levels, though in a region-specific manner. This is naturally important for studies aiming to develop therapies for the elderly.     

Retinal dopaminergic neurons (A17) expressing neuromedin K receptor (NK3): a double immunocytochemical study in the rat

By using a double immunofluorescence method we examined the distribution of dopaminergic neurons (A17) expressing neuromedin K receptor (NKR, NK₃) in the rat retina. The distribution of NKR-like immunoreactive (-LI) neurons partially overlapped that of tyrosine hydroxylase (TH)-LI neurons in the inner retina of section and flat-mount preparation. Neurons showing both TH- and NKR-like immunoreactivities were found in the retina (A17): 100% of these TH-LI neurons displayed NKR-like immunoreactivity, and they constituted about 3.5% of total NKR-LI neurons. The majority of double-labeled neurons with TH- and NKR-like immunoreactivities were distributed in the proximal inner nuclear layer and the upper part of inner plexiform layer of the retina, and characterized with appearance of amacrine cells. The present study has provided morphological evidence for direct physiological modulation of dopaminergic neurons by tachykinins through NKR in the rat retina (A17).     

Morphology of neurons in the basal forebrain nuclei of the rat: a Golgi study

The neuronal cell types and their morphology in the nucleus basalis (NB), in the horizontal and vertical limbs of the diagonal band of Broca (NHL and NVL), and in the medial septal nucleus (MSN) were examined in Golgi-impregnated material. Cells appeared as multipolar or oligopolar and displayed a variable dendritic morphology; their somata varied considerably both in shape and size. The dendrites of most cells were restricted within nuclear boundaries, although occasionally neurons located near boundaries, particularly cells in NHL, extended dendritic arbors into neighboring areas. Axons were rarely seen, but when they were found they were generally not impregnated beyond the initial segment and displayed no apparent preferential direction. Three types of cells common to each of the 4 nuclear groups could be identified on the basis of soma shape and dendritic form. The first type included large multipolar neurons with triangular or polygonal perikarya and typically 3-5 dendrites emerging from the poles of each cell. These cells were especially numerous in NB, NHL, and NVL, but were much less frequent in MSN. The second type comprised medium-sized cells with round or oval somata and a small number, usually 2-3, of dendrites. They constituted a large percentage of the cell population in MSN, but were also encountered in NHL and NVL as well as in NB. The third type consisted of cells with fusiform or spindle-shaped somata with usually single dendrites emanating from each pole of the cell. A rare but distinct type of spindle-shaped neuron with dendrites bearing a rich complement of long and thin appendages was observed mainly in the ventral border of NHL. The present observations suggest that although the proportions and sizes of the 3 types of neurons vary between the 4 nuclei, neurons throughout the basal forebrain share common morphological characteristics.     

Sparing of striatal neurons coexpressing calretinin and substance P (NK1) receptor in Huntington''s disease

Immunohistochemical studies of the striatum in normal human subjects with a double-antigen localization method have revealed the presence of large and medium-sized aspiny neurons displaying immunoreactivity for both the calcium-binding protein calretinin and substance P (neurokinin-1) receptor. These large and medium-sized cells form two distinct classes of striatal interneurons, which together represent less than 3% of the total neuronal population of the human striatum. Observations made in four cases of Huntington's disease revealed that such doubly labeled interneurons are still present in the striatum of these patients, despite the marked atrophy of the structure. This study provides the first evidence for the existence of interneurons containing calretinin and expressing tachykinin receptors in the human striatum. It also demonstrates the selective sparing of these chemospecific striatal neurons in Huntington's disease.     

Immunocytochemical evidence for the existence of substance P receptor (NK1) in serotonin neurons of rat and mouse dorsal raphe nucleus

In addition to its neurotransmitter/modulator role in pain perception, substance P (SP) is involved in a regulation of mood, as antagonists of its neurokinin-1 receptor (NK1r) have been found to have antidepressant-like effects in humans. In rodents, treatment with NK1r antagonists has been shown to increase the firing of dorsal raphe nucleus (DRN) serotonin (5-hydroxytryptamine, 5-HT) neurons and to induce a desensitization of their 5-HT1A autoreceptors, suggesting local interactions between the SP and 5-HT systems. To search for the presence of NK1r on 5-HT neurons of the DRN, we used light and electron microscopic immunocytochemistry, as well as confocal microscopy, after single- and double-labelling of NK1r and of the biosynthetic enzyme of 5-HT, tryptophan hydroxylase (TpOH). A significant number of 5-HT (TpOH-positive) cell bodies and dendrites endowed with NK1r were thus demonstrated in the caudal part of rat and mouse DRN. As visualized by electron microscopy after gold immunolabelling, NK1r was mostly cytoplasmic in 5-HT neurons, while predominating on the plasma membrane in the case of TpOH-negative dendrites. The proportion of NK1r observed on the plasma membrane of 5-HT neurons was, however, slightly higher in mouse than rat. Thus, in both rat and mouse DRN, a subpopulation of 5-HT neurons is endowed with NK1r receptors and may be directly involved in the antidepressant-like effects of NK1r antagonists. These 5-HT neurons represent a new element in the neuronal circuitry currently proposed to account for the role of SP in mood regulation.     

Substance P (neurokinin-1) receptor mRNA is selectively expressed in cholinergic neurons in the striatum and basal forebrain

In the striatum substance P (neurokinin-1) receptor, mRNA is selectively localized in large neurons that also express mRNA encoding choline acetyltransferase (ChAT) by in situ hybridization histochemistry. Substance P receptor mRNA is also localized in ChAT mRNA-containing neurons in the medial septum and basal forebrain cell groups. Thus, in the rat forebrain the substance P receptor appears to be expressed selectively by cholinergic neurons. Striatal neurons that contain substance P also utilize γ-aminobutyric acid (GABA) as a transmitter. These neurons make synaptic contact with striatal cholinergic neurons, which are shown here to express the substance P receptor, and with other GABAergic neurons in the striatum and substantia nigra, which express GABA receptors but not substance P receptors. This suggests that individual striatal neurons may differentially affect target neurons dependent on the receptors expressed by those target neurons.     

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.     

Collateral projection of substance P receptor expressing neurons in the medullary dorsal horn to bilateral parabrachial nuclei of the rat

Employing a combination of fluorescent retrograde double labeling and immunofluorescence histochemistry for substance P receptor (SPR), we examined the collateral projection from single SPR-like immunoreactive neurons in the medullary dorsal horn (caudal subnucleus of the spinal trigeminal nucleus) to bilateral parabrachial nuclei in the rat. After injection of fast blue (FB) or diamidino yellow (DY) into the right or left parabrachial nucleus, respectively, single-labeled FB or DY neurons and double-labeled FB/DY neurons were observed mainly bilaterally in laminae I and II of the medullary dorsal horn. Some of the single-labeled FB or DY and double-labeled FB/DY neurons showed SPR-like immunoreactivity, especially in lamina I. In lamina I of left medullary dorsal horn which is ipsilateral to the DY injection into the PBN, the percentages of double-labeled FB/DY neurons to the total number of FB- or DY-labeled neurons were 34.0% or 20.2%, triple-labeled FB/DY/SPR neurons to the total number of FB/DY double-labeled or SPR-like immunoreactive neurons were 22.0% or 2.4%, respectively. In lamina I of the right medullary dorsal horn which is ipsilateral to the FB injection into the PBN, the percentages of double-labeled FB/DY neurons to the total number of FB- or DY-labeled neurons were 12.9% or 59.3%, triple-labeled FB/DY/SPR neurons to the total number of double-labeled FB/DY or SPR-like immunoreactive neurons were 24. 6% or 3.9%, respectively. The results suggest that some of the single SPR expressing neurons in the medullary dorsal horn might be innervated by substance P containing primary afferent fibers and transmit sensory information diffusely to bilateral parabrachial nuclei by way of their axonal collaterals.     

Cholinergic limbic projections and behavioral role of basal forebrain nuclei in the rat

The purposes of the present study were to identify cholinergic non-neocortical projections of the basal forebrain and to determine the role of this region in the regulation of estrogen-dependent reproductive behaviors in the rat. Bilateral electrolytic lesions were placed in an area encompassing the horizontal limb of the diagonal band, as well as portions of the substantia innominata and magnocellular preoptic nucleus, and choline acetyltransferase (CAT) activity was assayed in microdissected brain areas seven days after lesion. Compared to sham surgery, lesions of this region significantly reduced CAT activity in the basal amygdala (34%), dorsal hippocampus (14%), cingulate cortex (25%), piriform cortex (36%), and entorhinal cortex (34%). Other limbic and midbrain structures do not appear to receive significant cholinergic innervation from this locus since no reductions in CAT were detected after bilateral lesions. These included the anterior hypothalamus, ventromedial hypothalamus, mammillary nucleus, habenula, subiculum, ventral hippocampus, insular cortex, central gray, and interpeduncular nucleus. Behaviorally, female rats with bilateral lesions of the basal forebrain displayed an unusually high incidence of rejection behavior in response to attempted mounts by stimulus male rats in sexual behavior tests. There was no effect of basal forebrain lesions on the incidence of lordosis exhibited by these females. The dissociation of rejection and lordosis suggests that distinct neural pathways mediate the occurrence of these reproductive behaviors and that rejection behavior may be regulated by basal forebrain pathways.     

Action of substance P (neurokinin-1) receptor activation on rat neostriatal projection neurons.

Substance P (SP) acts as a neurotransmitter in the neostriatum through the axon collaterals of spiny projection neurons. However, possible direct or indirect actions of SP on the neostriatal output neurons have not been described. Targets of SP terminals within the neostriatum include interneurons, spiny neurons, afferent fibers and boutons. SP induces the release of both dopamine (DA) and acetylcholine (ACh). Since some postsynaptic actions of both DA and ACh on spiny neurons are known, we asked if activation of neostriatal NK1-class receptors is able to reproduce them. The SP NK1-receptor agonist, GR73632 (1 microM), had both excitatory and inhibitory actions on virtually all spiny neurons tested at resting potential. The excitatory action was blocked by atropine and coursed with an increase in firing rate and input resistance (R(N)). The inhibitory action was blocked by haloperidol and coursed with a reduction in firing rate and R(N). Therefore, the release of both DA and ACh induced by NK1-receptor activation modulates indirectly the excitability of the projection neurons. SP facilitates the actions of these transmitters on the spiny neuron. A residual excitatory response to the NK1-receptor agonist was observed in 30% of a sample of neurons tested in the presence of both haloperidol and atropine. The increase in R(N) that accompanied this response could be observed in the presence of 1 microM TTX or 100 microM Cd2+, suggesting a direct effect. Double labeling showed that only SP-immunoreactive neurons were facilitated by NK1-receptor activation in these conditions.     

Cholinergic neurons in the basal forebrain of the cat have direct projections to the sensorimotor cortex

Previous studies showed that projections from the cells in the substantia innominata to the sensorimotor cortex exist in the monkey and rat and that this group of cortical afferent fibers utilizes acetylcholine as a neurotransmitter. Because similar studies have not been made in the cat, the following study was undertaken to analyze the extent and distribution of this projection from the basal forebrain. Horseradish peroxidase (HRP) injections were made into the anterior and posterior sigmoid gyri 24 h before, and diisopropyl fluorophosphate was injected intramuscularly 3 h before the anesthetized cats were perfused. Brain sections from these cats were incubated in solutions to simultaneously detect retrogradely transported HRP and acetylcholinesterase (AChE) reaction product within the somata. The cell bodies that contained reaction product for both HRP and AChE were mostly large (25 to 30 μm) and multipolar. These double-labeled cells were located in (i) the nucleus of the diagonal band in rostral sections, (ii) the globus pallidus, entopeduncular nucleus, and substantia innominata at the level of the anterior commissure, and (iii) the lateral hypothalamus in the most caudal sections. Many of these sites corresponded to that for the basal nucleus of Meynert, an aggregation of large multipolar neurons scattered throughout the basal forebrain. Although the presence of AChE within a cell does not define a cholinergic neuron, recent studies indicated that its presence is a requirement for this neurotransmitter. These data together with biochemical and immunocytochemical data indicate that a cholinergic projection to the sensorimotor cortex of cats arises from the basal forebrain. This pathway may play a vital role in memory and cognition.     

Cholinergic neurons of the basal forebrain mediate biochemical and electrophysiological mechanisms underlying sleep homeostasis

The tight coordination of biochemical and electrophysiological mechanisms underlies the homeostatic sleep pressure (HSP) produced by sleep deprivation (SD). We have reported that during SD the levels of inducible nitric oxide synthase (iNOS), extracellular nitric oxide (NO), adenosine [AD]_ex, lactate [Lac]_ex and pyruvate [Pyr]_ex increase in the basal forebrain (BF). However, it is not clear whether all of them contribute to HSP leading to increased electroencephalogram (EEG) delta activity during non‐rapid eye movement (NREM) recovery sleep (RS) following SD. Previously, we showed that NREM delta increase evident during RS depends on the presence of BF cholinergic (ChBF) neurons. Here, we investigated the role of ChBF cells in coordination of biochemical and EEG changes seen during SD and RS in the rat. Increases in low‐theta power (5–7 Hz), but not high‐theta (7–9 Hz), during SD correlated with the increase in NREM delta power during RS, and with the changes in nitrate/nitrite [NO_x]_ex and [AD]_ex. Lesions of ChBF cells using IgG 192‐saporin prevented increases in [NO_x]_ex, [AD]_ex and low‐theta activity, during SD, but did not prevent increases in [Lac]_ex and [Pyr]_ex. Infusion of NO donor DETA NONOate into the saporin‐treated BF failed to increase NREM RS and delta power, suggesting ChBF cells are important for mediating NO homeostatic effects. Finally, SD‐induced iNOS was mostly expressed in ChBF cells, and the intensity of iNOS induction correlated with the increase in low‐theta activity. Together, our data indicate ChBF cells are important in regulating the biochemical and EEG mechanisms that contribute to HSP.