Dynorphin inhibits basal forebrain cholinergic neurons by pre- and postsynaptic mechanisms.

KEY POINTS: The basal forebrain is an important component of the ascending arousal system and may be a key site through which the orexin neurons promote arousal. It has long been known that orexin-A and -B excite basal forebrain cholinergic neurons, but orexin-producing neurons also make the inhibitory peptide dynorphin. Using whole-cell recordings in brain slices, we found that dynorphin-A directly inhibits basal forebrain cholinergic neurons via κ-opioid receptors, and decreases afferent excitatory synaptic input to these neurons. While the effects of dynorphin-A and orexin-A desensitize over multiple applications, co-application of dynorphin-A and orexin-A produces a sustained response that reverses depending on the membrane potential of basal forebrain cholinergic neurons. At -40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at -70 mV the excitatory response to orexin-A prevails. ABSTRACT: The basal forebrain (BF) is an essential component of the ascending arousal systems and may be a key site through which the orexin (also known as hypocretin) neurons drive arousal and promote the maintenance of normal wakefulness. All orexin neurons also make dynorphin, and nearly all brain regions innervated by the orexin neurons express kappa opiate receptors, the main receptor for dynorphin. This is remarkable because orexin excites target neurons including BF neurons, but dynorphin has inhibitory effects. We identified the sources of dynorphin input to the magnocellular preoptic nucleus and substantia innominata (MCPO/SI) in mice and determined the effects of dynorphin-A on MCPO/SI cholinergic neurons using patch-clamp recordings in brain slices. We found that the orexin neurons are the main source of dynorphin input to the MCPO/SI region, and dynorphin-A inhibits MCPO/SI cholinergic neurons through κ-opioid receptors by (1) activation of a G protein-coupled inwardly rectifying potassium current, (2) inhibition of a voltage-gated Ca(2+) current and (3) presynaptic depression of the glutamatergic input to these neurons. The responses both to dynorphin-A and to orexin-A desensitize, but co-application of dynorphin-A and orexin-A produces a sustained response. In addition, the polarity of the response to the co-application depends on the membrane potential of BF neurons; at -40 mV the net effect of the co-application is inhibition by dynorphin-A, whereas at -70 mV the excitatory response to orexin-A prevails. This suggests that depending on their state of activation, BF cholinergic neurons can be excited or inhibited by signals from the orexin neurons.

GABAergic neurons expressing p75 in rat substantia innominata and nucleus basalis.

In vitro findings suggested a role for the p75 neurotrophin receptor in the maturation of GABAergic neurons residing in the basal forebrain (BF), a brain area known to have p75 expression only on cholinergic neurons. We document here the presence of GABAergic neurons which express p75 in the BF in vivo. Colocalization of p75 with the cholinergic marker choline-acetyltransferase (ChAT) and/or the GABAergic marker glutamic acid decarboxylase-67 (GAD67) was investigated in the BF at birth, at two weeks, and in adulthood. A subset of GAD67(+) neurons was p75(+) (p75(+)/GAD67(+)) but ChAT(-) in the substantia innominata and nucleus basalis magnocellularis at birth, whereas all p75(+)/GAD67(+) neurons were also ChAT(+) from two weeks onward. These phenotypic features suggest that a subpopulation of GABAergic neurons could be sensitive to neurotrophins during brain maturation. To unravel this issue, we then pursued a functional analysis by assessing p75 expression profile, and its modulation by nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) in primary BF cell cultures. NGF increased p75 expression exclusively in cholinergic neurons, whereas BDNF induced p75 expression only in a subset of GABAergic neurons (p75(+)/GAD67(+)/ChAT(-)) through a p75- and tyrosine-kinase-dependent mechanism. The latter findings point to a selective role of BDNF in the induction of p75 expression in BF GABAergic neurons. Altogether these results confirm the role of neurotrophins in the developing and mature circuitry of GABAergic neurons in the BF regions.

Effects of ibotenate and 192IgG-saporin lesions of the nucleus basalis magnocellularis/substantia innominata on spontaneous sleep and wake states and on recovery sleep after sleep deprivation in rats.

The basal forebrain (BF) is known for its role in cortical and behavioral activation, and has been postulated to have a role in compensatory mechanisms after sleep loss. However, specific neuronal phenotypes responsible for these roles are unclear. We investigated the effects of ibotenate (IBO) and 192IgG-saporin (SAP) lesions of the caudal BF on spontaneous sleep-waking and electroencephalogram (EEG), and recovery sleep and EEG after 6 h of sleep deprivation (SD). Relative to artificial CSF (ACSF) controls, IBO injections decreased parvalbumin and cholinergic neurons in the caudal BF by 43 and 21%, respectively, and cortical acetylcholinesterase staining by 41%. SAP injections nonsignificantly decreased parvalbumin neurons by 11%, but significantly decreased cholinergic neurons by 69% and cortical acetylcholinesterase by 84%. IBO lesions had no effect on sleep-wake states but increased baseline delta power in all states [up to 62% increase during non-rapid eye movement (NREM) sleep]. SAP lesions transiently increased NREM sleep by 13%, predominantly during the dark phase, with no effect on EEG. During the first 12 h after SD, animals with IBO and SAP lesions showed lesser rebound NREM sleep (32 and 77% less, respectively) and delta power (78 and 53% less) relative to ACSF controls. These results suggest that noncholinergic BF neurons promote cortical activation by inhibiting delta waves, whereas cholinergic BF neurons play a nonexclusive role in promoting wake. Intriguingly, these results also suggest that both types of BF neurons play important roles, probably through different mechanisms, in increased NREM sleep and EEG delta power after sleep loss.

Selective anterograde tracing of nonserotonergic projections from dorsal raphe nucleus to the basal forebrain and extended amygdala.

The dorsal raphe nucleus (DRN) contains both serotonergic and nonserotonergic projection neurons. Retrograde tracing studies have demonstrated that components of the basal forebrain and extended amygdala are targeted heavily by input from nonserotonergic DRN neurons. The object of this investigation was to examine the terminal distribution of nonserotonergic DRN projections in the basal forebrain and extended amygdala, using a technique that allows selective anterograde tracing of nonserotonergic DRN projections. To trace nonserotonergic DRN projections, animals were pretreated with nomifensine, desipramine and the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), 7 days prior to placing an iontophoretic injection of biotinylated dextran amine (BDA) into the DRN. In animals treated with 5,7-DHT, numerous nonserotonergic BDA-labeled fibers ascended to the basal forebrain in the medial forebrain bundle system. Some of these labeled fibers crossed through the lateral hypothalamus, bed nucleus of the stria terminalis, and substantial innominata. These fibers entered the amygdala through the ansa peduncularis and ramified within the central and basolateral amygdaloid nuclei. Other fibers entered the diagonal band of Broca and formed a dense plexus of labeled fibers in the dorsal half of the intermediate portion of the lateral septal nucleus and the septohippocampal nucleus. These findings demonstrate that the basal forebrain and extended amygdala receive a dense projection from nonserotonergic DRN neurons. Given that the basal forebrain plays a critical role in processes such as motivation, affect, and behavioral control, these findings support the hypothesis that nonserotonergic DRN projections may exert substantial modulatory control over emotional and motivational functions.

Different roles for amygdala central nucleus and substantia innominata in the surprise-induced enhancement of learning.

Within most modern learning theories, the discrepancy between expected and obtained outcomes ("prediction error" or "surprise") is a critical determinant of the acquisition of learned associations. The results of studies from many laboratories show that the surprising omission of an expected event may enhance attention to stimuli that remain present, such that subsequent learning about those stimuli is enhanced. A series of reports from our laboratories demonstrated that these surprise-induced enhancements of stimulus associability depend on circuitry that includes the amygdala central nucleus (CeA), the cholinergic neurons in the sublenticular substantia innominata/nucleus basalis magnocellularis (SI/nBM), as well as certain cortical projections of these latter neurons. In this study, we found very different roles for CeA and SI/nBM in surprise-induced enhancements of stimulus associability. In four experiments that used transient inactivation techniques, we found that surprise-induced enhancement of subsequent learning about a stimulus depended on intact CeA function at the time of surprise but not when more rapid learning was subsequently expressed. In contrast, normal SI/nBM function was critical to the expression of enhanced learning but was not necessary when surprise was induced. These data suggest that these two components of the so-called "extended amygdala" serve distinct roles in the encoding and retrieval of information used in modulating attention to stimuli in associative learning. Additional circuitry linking these brain regions may also be important in the maintenance of that information.

A Central Amygdala-Substantia Innominata Neural Circuitry Encodes Aversive Reinforcement Signals.

Aversive stimuli can impact motivation and support associative learning as reinforcers. However, the neural circuitry underlying the processing of aversive reinforcers has not been elucidated. Here, we report that a subpopulation of central amygdala (CeA) GABAergic neurons expressing protein kinase C-delta (PKC-δ+) displays robust responses to aversive stimuli during negative reinforcement learning. Importantly, projections from PKC-δ+ neurons of the CeA to the substantia innominata (SI) could bi-directionally modulate negative reinforcement learning. Moreover, consistent with the idea that SI-projecting PKC-δ+ neurons of the CeA encode aversive information, optogenetic activation of this pathway produces conditioned place aversion, a behavior prevented by simultaneous ablating of SI glutamatergic neurons. Taken together, our data define a cell-type-specific neural circuitry modulating associative learning by encoding aversive reinforcement signals.

Vesicular glutamate (VGlut), GABA (VGAT), and acetylcholine (VACht) transporters in basal forebrain axon terminals innervating the lateral hypothalamus.

The basal forebrain (BF) is known to play important roles in cortical activation and sleep, which are likely mediated by chemically differentiated cell groups including cholinergic, gamma-aminobutyric acid (GABA)ergic and other unidentified neurons. One important target of these cells is the lateral hypothalamus (LH), which is critical for arousal and the maintenance of wakefulness. To determine whether chemically specific BF neurons provide an innervation to the LH, we employed anterograde transport of 10,000 MW biotinylated dextran amine (BDA) together with immunohistochemical staining of the vesicular transporter proteins (VTPs) for glutamate (VGluT1, -2, and -3), GABA (VGAT), or acetylcholine (ACh, VAChT). In addition, we applied triple staining for the postsynaptic proteins (PSPs), PSD-95 with VGluT or Gephyrin (Geph) with VGAT, to examine whether the BDA-labeled varicosities may form excitatory or inhibitory synapses in the LH. Axons originating from BDA-labeled neurons in the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) descended within the medial forebrain bundle and extended collateral varicose fibers to contact LH neurons. In the LH, the BDA-labeled varicosities were immunopositive (+) for VAChT ( approximately 10%), VGluT2 ( approximately 25%), or VGAT ( approximately 50%), revealing an important influence of newly identified glutamatergic together with GABAergic BF inputs. Moreover, in confocal microscopy, VGluT2+ and VGAT+ terminals were apposed to PSD-95+ and Geph+ profiles respectively, indicating that they formed synaptic contacts with LH neurons. The important inputs from glutamatergic and GABAergic BF cells could thus regulate LH neurons in an opposing manner to stimulate vs. suppress cortical activation and behavioral arousal reciprocally.

Stereological estimates of the basal forebrain cell population in the rat, including neurons containing choline acetyltransferase, glutamic acid decarboxylase or phosphate-activated glutaminase and colocalizing vesicular glutamate transporters.

The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and comprise GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as approximately 355,000 and the numbers of ChAT-immuno-positive (+) as approximately 22,000, GAD+ approximately 119,000 and PAG+ approximately 316,000, corresponding to approximately 5%, approximately 35% and approximately 90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also has the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, vesicular glutamate transporter (VGluT) 3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or dendrites.

Adenosine inhibits basal forebrain cholinergic and noncholinergic neurons in vitro.

Adenosine has been proposed as a homeostatic "sleep factor" that promotes the transition from waking to sleep by affecting several sleep-wake regulatory systems. In the basal forebrain, adenosine accumulates during wakefulness and, when locally applied, suppresses neuronal activity and promotes sleep. However, the neuronal phenotype mediating these effects is unknown. We used whole-cell patch-clamp recordings in in vitro rat brain slices to investigate the effect of adenosine on identified cholinergic and noncholinergic neurons of the magnocellular preoptic nucleus and substantia innominata. Adenosine (0.5-100 microM) reduced the magnocellular preoptic nucleus and substantia innominata cholinergic neuronal firing rate by activating an inwardly rectifying potassium current that reversed at -82 mV and was blocked by barium (100 microM). Application of the A1 receptor antagonist 8-cyclo-pentyl-theophylline (200 nM) blocked the effects of adenosine. Adenosine was also tested on two groups of electrophysiologically distinct noncholinergic magnocellular preoptic nucleus and substantia innominata neurons. In the first group adenosine, via activation of postsynaptic A1 receptors, reduced spontaneous firing via inhibition of the hyperpolarization-activated cation current. Blocking the H-current with ZD7288 (20 microM) abolished adenosine effects on these neurons. The second group was not affected by adenosine. These results demonstrate that, in the magnocellular preoptic nucleus and substantia innominata region of the basal forebrain, adenosine inhibits both cholinergic neurons and a subset of noncholinergic neurons. Both of these effects occur via postsynaptic A1 receptors, but are mediated downstream by two separate mechanisms.

Sensitized attentional performance and Fos-immunoreactive cholinergic neurons in the basal forebrain of amphetamine-pretreated rats.

BACKGROUND: The consequences of repeated exposure to psychostimulants have been hypothesized to model aspects of schizophrenia. This experiment assessed the consequences of the administration of an escalating dosing regimen of amphetamine (AMPH) on attentional performance. Fos-like immunoreactivity (Fos-IR) in selected regions of these rats' brains was examined to test the hypothesis that AMPH-sensitized attentional impairments are associated with increased recruitment of basal forebrain cholinergic neurons. METHODS: Rats were trained in a sustained attention task and then treated with saline or in accordance with an escalating dosing regimen of AMPH (1-10 mg/kg). Performance was assessed during the pretreatment and withdrawal periods and following the subsequent administration of AMPH "challenges" (.5, 1.0 mg/kg). Brain sections were double-immunostained to visualize Fos-IR and cholinergic neurons. RESULTS: Compared with the acute effects of AMPH, AMPH "challenges," administered over 2 months after the pretreatment was initiated, resulted in significant impairments in attentional performance. In AMPH-pretreated and -challenged animals, an increased number of Fos-IR neurons was observed in the basal forebrain. The majority of these neurons were cholinergic. CONCLUSIONS: The evidence supports the hypothesis that abnormally regulated cortical cholinergic inputs represent an integral component of neuronal models of the attentional dysfunctions of schizophrenia.

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.

Separate sets of neurons of the central nucleus of the amygdala project to the substantia innominata and the caudal pontine reticular nucleus in the rat.

The central nucleus of the amygdala (CeA) is generally regarded as a control nucleus of subcortical target systems. Due to its widespread projections to different brain areas it is able to modulate emotional behavior of the organism. However, it is still not clear whether single neurons of the CeA project to different areas or to one target area. Injections of the retrograde tracers Fluorogold and True Blue into target regions of the central nucleus of the amygdala, i.e., the substantia innominata (SI) and the caudal pontine reticular nucleus (PNC), revealed overlapping but otherwise distinct neuronal populations within mainly the medial division of the CeA. From our study we conclude that SI and PNC receive input from different subsets of amygdala neurons.

Topography of the magnocellular basal forebrain system in human brain.

In primates, the large neurons in the nucleus basalis of Meynert (nbM), nucleus of the diagonal band of Broca (dbB), and medial septum are part of a cholinergic system with direct projections to amygdala, hippocampus, and cortex. Recent evidence indicates that neurons of this system selectively degenerate in individuals with Alzheimer's disease (AD) and suggests that degeneration of these cells contributes to the loss of presynaptic cortical cholinergic markers which occurs in these patients. The present report describes the topographical distribution of these large intensely basophilic, basal forebrain neurons in human brain. Rostrally, neurons of this magnocellular system are present in the medial septum and the dorsal and ventral parts of the nucleus of the dbB. The largest number occur in the nbM, which is situated in the substantia innominata below the globus pallidus. Caudally, large nbM-type neurons are found along the ventral and lateral edges of the globus pallidus. Neurons of this type are also encountered in the white matter below the putamen and nucleus accumbens, at the edges of the anterio commissure, in the white matter laminae of the globus pallidus and within and at the medial edge of the genu of the interal capsule. Directions for dissection of this system in human brain are given in an Appendix.

Stability of neuronal number in the human nucleus basalis of Meynert with age.

Numbers of neurons in the nucleus basalis of Meynert were estimated in seventeen non-demented patients who died of chronic hepatic or cardiopulmonary disease. Neurons were counted at the site of maximal neuronal density (SMND). This site was chosen by reviewing serial sections around the decussation of the anterior commissure and appeared to be comparable in different individuals. No correlation between numbers of neurons and age could be found. It appears that no uniform neuronal loss occurs in the nucleus basalis with age. Taken together with biochemical studies of cerebral cortical choline acetyltransferase activity, these findings suggest that there is no overall change in cholinergic input to cerebral cortex with age.

Stability of numbers but not size of mouse forebrain cholinergic neurons to 53 months.

In normal mammalian aging there is a reduction of cholinergic markers in a variety of regions. To determine whether this reduction is related to reduced numbers of basal forebrain cholinergic neurons, we counted the number and measured the sizes of the magnocellular acetylcholinesterase-positive neurons in this region of 7, 15, and 53-month-old C57Bl/6NNIA mice. Data were collected from coded slides containing the medial septum, nucleus of the diagonal band, magnocellular preoptic nucleus, and nucleus basalis magnocellularis. There was no decline in numbers of basal forebrain acetylcholinesterase-positive neurons in any of the regions studied. However, cell sizes showed a progressive age-related decline which was greatest in the nucleus basalis magnocellularis.

Morphological and electrophysiological characteristics of noncholinergic basal forebrain neurons.

Cholinergic neurons in the basal forebrain are the focus of considerable interest because they are severely affected in Alzheimer's disease. However, both cholinergic and noncholinergic neurons are intermingled in this region. The goal of the present study was to characterize the morphology and in vivo electrophysiology of noncholinergic basal forebrain neurons. Neurons in the ventral pallidum and substantia innominata were recorded extracellularly, labeled juxtacellularly with biocytin and characterized for the presence of choline acetyltransferase immunoreactivity. Two types of ventral pallidal cells were observed. Type I ventral pallidal neurons had axons that rarely branched near the cell body and tended to have smaller somata and lower spontaneous firing rates than did type II ventral pallidal neurons, which displayed extensive local axonal arborizations. Subtypes of substantia innominata neurons could not be distinguished based on axonal morphology. These noncholineregic neurons exhibited local axon arborizations along a continuum that varied from no local collaterals to quite extensive arbors. Substantia innominata neurons had lower spontaneous firing rates, more variable interspike intervals, and different spontaneous firing patterns than did type II ventral pallidal neurons and could be antidromically activated from cortex or substantia nigra, indicating that they were projection neurons. Ventral pallidal neurons resemble, both morphologically and electrophysiologically, previously described neurons in the globus pallidus, whereas the substantia innominata neurons bore similarities to isodendritic neurons of the reticular formation. These results demonstrate the heterogeneous nature of noncholinergic neurons in the basal forebrain.

Subset of neurons characterized by the presence of NADPH-diaphorase in human substantia innominata.

The substantia innominata encompasses an area of the basal forebrain that is ventral to the lenticular nucleus and anterior commissure, medial to the claustrum and external capsule, and lateral to the hypothalamus. The nucleus basalis of Meynert consists primarily of large acetylcholinesterase (AchE)-positive neurons embedded within the substantia innominata. Damage to these neurons may be important in the pathogenesis of cortical dysfunction in Alzheimer's disease. In order to characterize other neuronal elements in the substantia innominata and their relationship to the nucleus basalis, we chose to study a biochemically distinct neuronal subset containing the enzyme nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). The substantia innominata was blocked from six normal brains obtained postmortem and fixed in neutral-buffered formalin at 4 degrees C for 48 hours. Free-floating 50-micron sections from several levels were stained for NADPH-d or AchE activities. Selected sections were double stained for NADPH-d and AchE. NADPH-d activity was present in a network of pleomorphic neurons that extended through all levels of the substantia innominata and into the striatum and amygdala. NADPH-d neurons were particularly numerous at the level of the anterior commisure and were closely associated with the cholinergic neurons of the nucleus basalis. They were not seen in the ventral pallidum, or the vertical limb of the diagonal band of Broca or in the islands of Calleja. The cell bodies of NADPH-d neurons were quite varied in shape, ranging from ovoid to fusiform, and about half the cells were bipolar. Where neuronal density was high, their dendrites formed an interlacing pattern. NADPH-d-positive fibres were seen coursing through the external capsule, hypothalamus, and amygdala. This novel set of neurons in the substantia innominata may be part of a more extensive network that interacts with the magnocellular basal forebrain system at the level of the nucleus basalis. Whether other neurotransmitters are present within these neurons and whether NADPH-d neurons are involved in Alzheimer's disease remain to be elucidated.

Efferent projections of the subthalamic nucleus in the squirrel monkey as studied by the PHA-L anterograde tracing method.

The organization of the efferent connections of the subthalamic nucleus was studied in the squirrel monkey (Saimiri sciureus) by using the lectin Phaseolus vulgaris-leucoagglutinin (PHA-L) as an anterograde tracer. At the level of the basal forebrain, anterogradely labeled fibers and axon terminals were mostly found in the striatopallidal complex and the substantia innominata. In cases in which the PHA-L injection sites were placed in the central or the lateral third of the subthalamic nucleus, numerous anterogradely labeled fibers were seen to arise from the injection loci and innervate massively the globus pallidus. At pallidal levels the fibers formed bands lying parallel and adjacent to the medullary laminae. The number and the complexity of the topographical organization of these bands varied with the size and the location of the PHA-L injection site. When examined at a higher magnification, the bands of subthalamopallidal fibers appeared as rich plexuses of short axon collaterals with small bulbous enlargements that closely surrounded the cell bodies and primary dendrites of pallidal cells. In contrast, PHA-L injection involving the medial tip of the subthalamic nucleus did not produce bandlike fiber patterns in the globus pallidus. Instead, the labeled fibers formed a diffuse plexus occupying the ventral part of the rostral pole of the globus pallidus as well as the subcommissural pallidal region. The substantia innominata contained a moderate number of labeled fibers and axon terminals following injection of PHA-L in the medial tip of the subthalamic nucleus. A small to moderate number of anterogradely labeled fibers were seen in the putamen after all PHA-L injections. These subthalamostriatal fibers were long, linear, and branched infrequently. At midbrain level the substantia nigra contained a significant number of anterogradely labeled fibers and axon terminals following PHA-L injection in the subthalamic nucleus. The subthalamonigral fibers descended along the ventromedial part of the cerebral peduncle and swept laterally to reach their target. Most of these fibers formed small plexuses along the base of the pars reticulata, whereas a few others ascended along the cell columns of the pars compacta that impinged deeply within the pars reticulata. More caudally in the brainstem, a small number of fibers occurred in the area of the pedunculopontine nucleus and in the periaqueductal gray. These findings indicate that besides its well-known connection with the pallidum, the subthalamic nucleus gives rise to widespread projections to other components of the basal ganglia in primates.

Distribution of beta-nerve growth factor receptors in the human basal forebrain.

The distribution of neurons expressing the receptor for beta-nerve growth factor has been examined immunohistochemically in serial coronal sections of basal forebrain from aged normal human subjects. Neurons expressing the receptor were observed in the nucleus of the diagonal band of Broca and in the anterior, the intermediate, and the posterior portions of the nucleus basalis of Meynert. Neurons could also be seen in the medial septal nucleus and embedded in myelinated fibre tracts such as those of the external capsule, cingulum, medullary laminae of the globus pallidus, ansa penduncularis, ansa lenticularis, and anterior commissure. In situ hybridization with a 35S cDNA probe to the human beta-nerve growth factor receptor confirms a neuronal location as the site of synthesis of beta-nerve growth factor receptors in the nucleus basalis of Meynert in a fifth brain. A high percentage of Nissl-stained hyperchromic magnocellular neurons expressed the receptor for beta-nerve growth factor, suggesting that most neurons in the human cholinergic magnocellular basal forebrain system express these receptors. Recent data suggest that beta-nerve growth factor functions as a neurotrophic factor in basal forebrain cholinergic neurons. In Alzheimer's disease there is known to be a reduction in cholinergic function and an apparent loss of neurons in the cholinergic nucleus basalis of Meynert. For this reason we have examined the distribution of receptors for beta-nerve growth factor in the normal human basal forebrain in order to form a basis for comparison to those with Alzheimer's disease.

Noradrenergic innervation of vasopressin- and oxytocin-containing neurons in the hypothalamic paraventricular nucleus of the macaque monkey: quantitative analysis using double-label immunohistochemistry and confocal laser microscopy.

Previous reports on the rat and monkey hypothalamus have revealed a dense noradrenergic innervation within the hypothalamic paraventricular nucleus as assessed by dopamine-beta-hydroxylase immunohistochemistry. These single-label analyses were unable to delineate the cellular structures which receive this catecholaminergic innervation. Double-label preparations in the rat hypothalamic paraventricular nucleus have demonstrated synaptic interactions between noradrenergic varicosities and magnocellular neurons. However, the density and distribution of varicosities contacting chemically identified magnocellular neurons have not been assessed at the light or electron microscopic level. In this report, single-label immunohistochemistry was used to assess the morphology and distribution of vasopressin- and oxytocin-immunoreactive neurons within the macaque hypothalamic paraventricular nucleus. In addition, double-label immunohistochemistry was combined with confocal laser scanning microscopy to quantify the number of dopamine-beta-hydroxylase-immunoreactive varicosities in apposition to magnocellular neurons expressing vasopressin or oxytocin immunoreactivity. The morphology of chemically identified neurons was also compared to magnocellular neurons in the monkey hypothalamic paraventricular nucleus which were filled with Lucifer Yellow in order to assess the somatodendritic labeling of the immunohistochemical preparation. Qualitative assessment of immunohistochemically identified magnocellular cells indicated that vasopressin- and oxytocin-containing neurons are observed throughout the rostrocaudal extent of the monkey hypothalamic paraventricular nucleus, demarcating this structure from the surrounding anterior hypothalamus. The distribution of the two nonapeptides is complementary, with vasopressin-immunoreactive neurons having a greater somal volume and located in a more medial aspect of the mid and caudal hypothalamic paraventricular nucleus relative to oxytocin-immunoreactive perikarya. For the double-label preparations, a series of confocal optical sections was assessed through the total somal volume of vasopressin- and oxytocin-immunoreactive neurons along with the corresponding dopamine-beta-hydroxylase-immunoreactive varicosities in the same volume of tissue, generating a varicosity-to-neuron ratio which was further characterized morphologically to assess afferent input to the soma and proximal dendrites. Quantitative analysis revealed that vasopressin-immunoreactive neurons received approximately two thirds of their dopamine-beta-hydroxylase-immunoreactive varicosities in apposition to the proximal dendrites and one third in apposition to the somata. Furthermore, vasopressin-immunoreactive neurons received a greater innervation density than oxytocin-immunoreactive neurons, which did not have a differential distribution of varicosities on the proximal dendrites and somata. The distribution of dopamine-beta-hydroxylase-immunoreactive afferents on magnocellular neurons in the hypothalamic paraventricular nucleus may reflect a physiological role of this circuit in terms of preferential release of vasopressin from magnocellular neurons upon noradrenergic stimulation.