Alterations in Sub-Axonal Architecture Between Normal Aging and Parkinson's Diseased Human Brains Using Label-Free Cryogenic X-ray Nanotomography.

Gaining insight to pathologically relevant processes in continuous volumes of unstained brain tissue is important for a better understanding of neurological diseases. Many pathological processes in neurodegenerative disorders affect myelinated axons, which are a critical part of the neuronal circuitry. Cryo ptychographic X-ray computed tomography in the multi-keV energy range is an emerging technology providing phase contrast at high sensitivity, allowing label-free and non-destructive three dimensional imaging of large continuous volumes of tissue, currently spanning up to 400,000 µm3. This aspect makes the technique especially attractive for imaging complex biological material, especially neuronal tissues, in combination with downstream optical or electron microscopy techniques. A further advantage is that dehydration, additional contrast staining, and destructive sectioning/milling are not required for imaging. We have developed a pipeline for cryo ptychographic X-ray tomography of relatively large, hydrated and unstained biological tissue volumes beyond what is typical for the X-ray imaging, using human brain tissue and combining the technique with complementary methods. We present four imaged volumes of a Parkinson's diseased human brain and five volumes from a non-diseased control human brain using cryo ptychographic X-ray tomography. In both cases, we distinguish neuromelanin-containing neurons, lipid and melanic pigment, blood vessels and red blood cells, and nuclei of other brain cells. In the diseased sample, we observed several swellings containing dense granular material resembling clustered vesicles between the myelin sheaths arising from the cytoplasm of the parent oligodendrocyte, rather than the axoplasm. We further investigated the pathological relevance of such swollen axons in adjacent tissue sections by immunofluorescence microscopy for phosphorylated alpha-synuclein combined with multispectral imaging. Since cryo ptychographic X-ray tomography is non-destructive, the large dataset volumes were used to guide further investigation of such swollen axons by correlative electron microscopy and immunogold labeling post X-ray imaging, a possibility demonstrated for the first time. Interestingly, we find that protein antigenicity and ultrastructure of the tissue are preserved after the X-ray measurement. As many pathological processes in neurodegeneration affect myelinated axons, our work sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues.

The blood clotting Factor XIIIa forms unique complexes with amyloid-beta (Aß) and colocalizes with deposited Aß in cerebral amyloid angiopathy.

AIMS: Cerebral amyloid angiopathy (CAA) is a key pathological hallmark of Alzheimer's disease (AD) characterized by accumulation of amyloid-beta (Aß) protein in blood vessel walls. CAA impairs vessel functioning, affects blood brain barrier integrity and accelerates cognitive decline of AD patients. Unfortunately, mechanisms underlying Aß deposition in the vessel wall remain largely unknown. Factor XIIIa (FXIIIa) is a blood-derived transglutaminase crucial in blood coagulation by cross-linking fibrin molecules. Evidence is mounting that blood-derived factors are present in CAA and may play a role in protein deposition in the vessel wall. We therefore investigated whether FXIIIa is present in CAA and if FXIIIa cross-link activity affects Aß aggregation. METHODS: Using immunohistochemistry, we investigated the distribution of FXIIIa, its activator thrombin and in situ FXIIIa activity in CAA in post-mortem AD tissue. We used surface plasmon resonance and Western blot analysis to study binding of FXIIIa to Aß and the formation of FXIIIa-Aß complexes, respectively. In addition, we studied cytotoxicity of FXIIIa-Aß complexes to cerebrovascular cells. RESULTS: FXIIIa, thrombin and in situ FXIIIa activity colocalize with the Aß deposition in CAA. Furthermore, FXIIIa binds to Aß with a higher binding affinity for Aß1-42 compared with Aß1-40 . Moreover, highly stable FXIIIa-Aß complexes are formed independently of FXIIIa cross-linking activity that protected cerebrovascular cells from Aß-induced toxicity in vitro. CONCLUSIONS: Our data showed that FXIIIa colocalizes with Aß in CAA and that FXIIIa forms unique protein complexes with Aß that might play an important role in Aß deposition and persistence in the vessel wall.

Anti-inflammatory effect by lentiviral-mediated overexpression of IL-10 or IL-1 receptor antagonist in rat glial cells and macrophages.

Neuroinflammation, as defined by activation of local glial cells and production of various inflammatory mediators, is an important feature of many neurological disorders. Expression of pro-inflammatory mediators produced by glial cells in the central nervous system (CNS) is considered to contribute to the neuropathology observed in those diseases. To diminish the production or action of pro-inflammatory mediators, we have used lentiviral (LV) vector-mediated encoding rat interleukin-10 (rIL-10) or rat interleukin-1 receptor antagonist (rIL-1ra) to direct the local, long-term expression of these anti-inflammatory cytokines in the CNS. We have shown that cultured macrophages or astroglia transduced with LV-rIL-10 or LV-rIL-1ra produced far less tumor necrosis factor (TNF)alpha or IL-6, respectively in response to pro-inflammatory stimuli. Moreover, intracerebroventricular (i.c.v.) administration of LV-rIL-10 or LV-rIL-1ra resulted in transduction of glial cells and macrophages and, subsequently reduced TNFalpha, IL-6 and inducible nitric oxide synthase (iNOS) expression in various brain regions induced by inflammatory stimuli, whereas peripheral expression of these mediators remained unaffected. In addition, expression levels of the anti-inflammatory cytokines IL-4 and transforming growth factor-beta were not altered in either brain or pituitary gland. Furthermore, i.c.v. administration of LV-rIL-10 or LV-rIL-1ra given during the remission phase of chronic-relapsing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, improved the clinical outcome of the relapse phase. Thus, local application of LV vectors expressing anti-inflammatory cytokines could be of therapeutic interest to counteract pro-inflammatory processes in the brain without interfering with the peripheral production of inflammatory mediators.

Changed distribution pattern of the constitutive rather than the inducible HSP70 chaperone in neuromelanin-containing neurones of the Parkinsonian midbrain.

Aberrant protein aggregation has been recognized as an important factor in the degeneration of melanized dopaminergic neurones in Parkinson's disease (PD). The constitutive (HSP73) and (heat)-inducible (HSP72) proteins of the heat shock 70 family form a major defence system against pathological protein aggregation. However, the distribution patterns of these chaperones in nigral neuromelanin-laden neurones are largely unknown. The present study determined the distribution of HSP72 and HSP73 in control and Parkinsonian substantia nigra, using immunohistochemistry. In the neuromelanin-laden neurones of controls, HSP72 was nondetectable, whereas HSP73 was weakly expressed in both the cytosol and the nucleus. Surprisingly, in PD subjects, marked nuclear HSP73, but not HSP72 immunoreactivity was observed, while cytosolic immunoreactivity of the two chaperones resembled the labelling pattern observed in controls. Furthermore, HSP73 immunoreactivity was observed in a subset of the Lewy bodies (LBs) detected in the substantia nigra of PD subjects, whereas only few of these LBs were labelled with HSP72. Interestingly, HSP72 and to a lesser extent HSP73 immunoreactivity was much stronger in nonmelanized neurones as compared with melanized neurones in this area. Thus, we conclude that the distribution pattern of HSP73 rather than HSP72 is changed in the nigral neuromelanin-laden neurones of PD subjects as compared with control subjects. The impaired ability of aged, dopaminergic neurones to express high levels of chaperones, may contribute to the preferential vulnerability of the latter cells in PD.

Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra.

Dopamine (DA) autooxidation, and consequent formation of neurotoxic DA-derived quinones and reactive oxygen species, has been implicated in dopaminergic cell death and, hence, in the pathogenesis of Parkinson's disease (PD). Stimulation of pathways involved in the detoxication of DA-quinones in the brain is hypothesized to be an effective means to limit oxidative stress and to confer neuroprotection in PD. In this respect, the inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) is of particular interest as it is directly implicated in the detoxication of DA-quinones and, in addition, has broad spectrum anti-oxidant properties. To study the potential pathophysiological role of NQO1 in PD, the cellular expression of NQO1 was examined in the mesencephalon of PD patients and age-matched controls. In the substantia nigra pars compacta (SNpc), NQO1 was found to be expressed in astroglial and endothelial cells and, albeit less frequently, also in dopaminergic neurons. Moreover, while overt NQO1 immunoreactivity was absent in the surrounding nervous tissue, in the Parkinsonian SNpc a marked increase in the astroglial and neuronal expression of NQO1 was consistently observed.

A single administration of interleukin-1 or amphetamine induces long-lasting increases in evoked noradrenaline release in the hypothalamus and sensitization of ACTH and corticosterone responses in rats.

Single administration of the cytokine interleukin-1beta (IL-1) or the psychostimulant amphetamine causes long-term sensitization of the hypothalamus pituitary adrenal (HPA) axis, i.e. enhanced adrenocorticotropine hormone (ACTH) and corticosterone responses weeks later. HPA responses to these stimuli involve activation of hypothalamic corticotropin-releasing hormone (CRH) neurons by noradrenergic projections to the paraventricular nucleus (PVN). In search of the underlying mechanisms, we studied the temporal pattern of HPA sensitization in relation to (1) the reactivity of noradrenergic projections to the PVN and (2) altered secretagogue production in hypothalamic CRH neurons. Single exposure to IL-1 or amphetamine induced cross-sensitization of ACTH and corticosterone responses 11 and 22 days later, but not after 42 days. Amphetamine-induced HPA sensitization was not accompanied by increased costorage of arginine vasopressin (AVP) in CRH terminals, as found previously after IL-1 pretreatment. The reactivity of noradrenergic terminals was assessed by measuring the electrically evoked release of [3H]-noradrenaline from superfused PVN slices. Single administration of amphetamine and IL-1 induced a long-lasting (up to 22 days) increase (up to 165%) of evoked noradrenaline release. This indicates that single exposure to psychostimulants or to cytokines can induce a long-lasting increase in stimulus-secretion coupling in brainstem noradrenergic neurons that innervate the PVN. This common, long-lasting functional change may underlie, at least in part, IL-1- and amphetamine-induced HPA cross-sensitization. In addition, increased AVP signalling by hypothalamic CRH neurons appears to play a role in IL-1-induced, but not in amphetamine-induced, HPA sensitization.

Vagotomy does not inhibit high dose lipopolysaccharide-induced interleukin-1beta immunoreactivity in rat brain and pituitary gland.

In the present study, we examined whether the vagus nerve is involved in mediating lipopolysaccharide (LPS)-induced appearance of IL-1beta immunoreactive cells in the brain and pituitary gland. Rats were either sham-operated or subjected to subdiaphragmatic vagotomy. Four weeks later, pyrogen free saline or 400 microg/kg LPS was administered to the rats intraperitoneally. Four and 8 h later, the animals were intracardially perfused with 4% paraformaldehyde and tissues were prepared for IL-1beta immunocytochemistry. IL-1beta positive cells were observed at both time-intervals after LPS administration in the choroid plexus, meninges, circumventricular organs and pituitary gland of both sham-operated and vagotomized rats. We conclude that under the conditions studied, the vagus nerve does not mediate LPS-induced appearance of IL-1beta in the rat brain and pituitary gland.

Immunohistochemical localization of interleukin-1beta, interleukin-1 receptor antagonist and interleukin-1beta converting enzyme/caspase-1 in the rat brain after peripheral administration of kainic acid.

The temporal and anatomical distribution of members of the interleukin-1 system in the rat brain following intraperitoneal kainic acid administration was studied in relation to neurodegeneration as detected with in situ end labelling. Kainic acid administration (10 mg/kg, i.p.) resulted in the induced expression of interleukin-1beta, interleukin- receptor antagonist and caspase-1p10 immunoreactivity in areas known to display neuronal and tissue damage upon excitotoxic lesions. The induction of these proteins was transient. Interleukin-1 immunoreactivity appeared at 5 h, and the interleukin-1 receptor antagonist-immunoreactive cells were first detected at 12 h, whereas the induction of caspase- 1p10 expression was first detected 24 h after kainic acid injection. Double labelling with the microglial marker Ox42 confirmed that both interleukin-1beta and interleukin-1 receptor antagonist were mainly localized in microglial cells. The regional distribution of in situ end-labelled neurons was similar to the distribution of cells expressing interleukin-1beta and interleukin-1 receptor antagonist, whereas the distribution of caspase-1 was more limited. The in situ end-labelled neurons, were, similarly to the interleukin-1beta-positive cells, first detected at 5 h, which is earlier than the induction of caspase-1. Our results show that the induction of IL-1beta and IL-1 receptor antagonist proteins after kainic acid are closely associated with the temporal as well as the anatomical distribution of in situ end-labelled neurons, whereas the induction of caspase-1 protein exhibited a delayed temporal profile and limited distribution. Since cytokine production occurs in activated microglial cells, the inflammatory component seems to be a strong mediator of this type of excitotoxic damage. The late onset of the caspase-1 expression would seem to indicate that this enzyme has no fundamental role in directly causing neuronal cell death induced by systemic kainic acid.

Bacterial endotoxin induces fos immunoreactivity in primary afferent neurons of the vagus nerve.

Subdiaphragmatic vagotomy inhibits brain-mediated illness responses to peripherally administered bacterial endotoxin, including fever, hyperalgesia, sickness behavior, and activation of the hypothalamic-pituitary-adrenal axis. However, direct evidence implicating vagal afferents specifically in conveying information about peripheral immune activation to the brain is still lacking. This study assessed whether (1) endotoxin induces the expression of the functional activation marker Fos in the vagal sensory ganglia, and (2) vagotomy abrogates endotoxin-induced Fos expression in these ganglia. Male rats, which had previously received vagotomy or sham surgery, were injected intraperitoneally or intravenously with either endotoxin or saline. Fos immunolabeling was absent in saline-treated rats. In contrast, scattered cells within the vagal sensory ganglia showed Fos immunoreactivity after both intraperitoneal and intravenous endotoxin administration in sham-operated rats. Vagotomy abolished Fos expression after intraperitoneal endotoxin administration, whereas after intravenous administration Fos expression was strongly attenuated, but not eliminated. These findings implicate vagal afferents as a potential signaling pathway to brain regions that generate illness responses to pro-inflammatory mediators.

Repeated administration of a selective dopamine D2 receptor agonist to 6-OHDA-lesioned rats does not affect the survival and outgrowth of intrastriatal fetal mesencephalic grafts.

The objective of the present study was to investigate the effects of chronic activation of dopamine D2 receptors on the development of grafted fetal rat mesencephalic dopaminergic neurons. Therefore, unilaterally 6-hydroxydopamine - lesioned rats received intrastriatal mesencephalic cell suspension grafts and were subsequently chronically treated with the selective dopamine D2 receptor agonist LY 171555 (Quinpirole). After treatment for 6 consecutive weeks, the rats were processed for tyrosine-hydroxylase immunocytochemistry to assess the survival and outgrowth from grafted dopaminergic neurons. morphological analysis revealed that, like the volume and morphology of the graft, neither the number nor the cell area of grafted dopaminergic neurons was significantly different between vehicle- and LY 171555-treated animals. To obtain a quantitative estimate of the graft-derived dopaminergic reinnervation, a computerized image analysis system was used. Using this procedure, which was based on the densitometric measurement of tyrosine hydroxylase immunoreactivity in the area adjacent to the grafted tissue, it was found that the extent of graft-derived outgrowth also appeared to be unaffected upon chronic treatment with LY 171555. It is concluded that long-term concurrent administration of a dopamine D2 receptor agonist for 6 consecutive weeks does not impair the survival and outgrowth of grafted rat fetal mesencephalic dopaminergic neurons.

Immunocytochemistry of cGMP in the Cerebellum of the Immature, Adult, and Aged Rat: the Involvement of Nitric Oxide. A Micropharmacological Study.

In this study we describe the localization of formaldehyde-fixed cGMP-immunoreactivity (cGMP-IR) in rat cerebellar tissue slices incubated in vitro. In the absence of phosphodiesterase inhibition, cGMP-immunofluorescence was of low intensity in tissue slices prepared from immature cerebella. Addition of isobutylmethylxanthine (IBMX) to the incubation medium resulted in the appearance of cGMP-IR in clusters of astrocytes in the internal granular layer. Addition of N-methyl-d-aspartate (NMDA), kainic acid, atrial natriuretic factor (ANF), or sodium nitroprusside (SNP) gave an intense cGMP-IR in Bergmann fibres, Bergmann cell bodies, and astrocytes in the internal granular layer. Astrocytes in the white matter showed cGMP-IR after incubation of the slice in the presence of ANF or nitroprusside, but not after NMDA or kainic acid. In addition, after SNP stimulation of cGMP production, cGMP-IR was found in fibres which were not positive for glial fibrillary acidic protein (GFAP). In the adult cerebellar slice, intense basal cGMP-immunostaining was observed in Bergmann fibres, Bergmann cell bodies, and astrocytes in the granular layer. No cGMP-IR was observed in Purkinje cells. Stimulation of the cGMP-content in the glial structures by NMDA, ANF, or SNP, was suggested by the immunocytochemical results. However, when measured biochemically, only the effect of SNP was statistically significant, and immunocytochemistry showed that SNP clearly stimulated cGMP synthesis in neuronal cell structures. In the cerebellum of the aged rat a reduced cGMP-IR was found compared to the adult, in the same structures which showed cGMP-IR in the adult. Basal cGMP-immunostaining was reduced in the presence of haemoglobin, methylene blue, by inhibiting nitric oxide synthesis with NG-monomethyl-l-arginine (NGMAr), or by depletion of external Ca2+. Also the stimulatory effect of NMDA and of ANF (partly) on the cGMP-IR was inhibited by these compounds. cGMP-IR after stimulation of guanylate cyclase by SNP was reduced by the concomitant presence of haemoglobin or methylene blue, but not by NGMAr, or by omission of Ca2+. Our results point to an important role for cGMP in the functioning of glial tissue in the cerebellum and also suggest a role for nitric oxide as an intercellular mediator in the functioning of glutamate and ANF in the cerebellum.

Localization of cGMP in the cerebellum of the adult rat: an immunohistochemical study.

The localization of cGMP in the cerebellum of the adult rat after fixation with formaldehyde was studied with an antibody raised against a cGMP-formaldehyde-thyroglobulin conjugate. Three different protocols were used: (1) in vitro incubation of 300 microns cerebellar slices followed by fixation and cryostat sectioning; (2) in vitro incubation of 100 microns cerebellar slices followed by fixation with no further sectioning; (3) perfusion fixation of the anesthetized rat followed by vibratome sectioning. All 3 protocols gave essentially the same results: cGMP-immunoreactivity was found predominantly in Bergmann fibers in the molecular layer, in Bergmann cell bodies in the Purkinje cell layer (but not in Purkinje cells), and in astroglial cells in the granular layer.

cGMP-Producing, Atrial Natriuretic Factor-Responding Cells in the Rat Brain.

Using an in vitro incubation method, we stimulated cGMP production in rat brain slices by rat ANF-(103 - 126). The localization of the cells responding to this ANF stimulation with an increase in cGMP production was studied by cGMP immunocytochemistry. ANF-responding cells were found in specific loci throughout the central nervous system of the rat. Regions containing the highest number of these cells were: the olfactory bulb, the lateral septum, the bed nucleus of the accessory olfactory tract, the mediobasal amygdala, the central grey area, the medial vestibular nucleus, and the nucleus of the solitary tract. Scattered ANF-responding, cGMP-immunoreactive cells were found in the hippocampus, the cingulate cortex, the ventral pallidum, the medial preoptic area, and the endopeduncular nucleus. ANF-responding cells in these areas had the same morphology, that is, multipolar with numerous processes. The nature of these ANF-responding cells was studied by sequential staining with an antiserum against glial fibrillary acidic protein (GFAP). In the hippocampus it was demonstrated that all ANF-responding cells are astroglial cells. However, not all astroglial cells in this area showed a cGMP response, demonstrating a regional heterogeneity. ANF-responding cells, having the appearance of neuronal cell bodies, could be found in the subfornical organ, and the hypothalamic paraventricular nucleus. Fibres producing cGMP immunoreactivity in response to ANF were found in the median preoptic nucleus, the medial preoptic area, and the dorsal hypothalamus.

Atrial natriuretic factor-responding and cyclic guanosine monophosphate (cGMP)-producing cells in the rat hippocampus: a combined micropharmacological and immunocytochemical approach.

Atrial natriuretic factor (ANF)-responding, cyclic guanosine monophosphate (cGMP)-producing cells were visualized in the hippocampus of the rat applying cGMP immunocytochemistry to hippocampal tissue slices incubated in vitro. These cells were found scattered throughout the hippocampus with their highest density in the hilus fascia dentata. Staining with an antibody against glial fibrillary acidic protein showed a completely different pattern, suggesting a non-glial nature of the ANF-responding cells. cGMP accumulation assayed biochemically was observed already at 10 microM ANF and was sensitive to inhibition by Methylene blue. The immunocytochemical data were fully supported by the biochemical measurements of cGMP under these conditions. Our results show for the first time a response to ANF of individual, cGMP-producing cells in the CNS of the rat.

Immunohistochemical localization of monoamines and cyclic nucleotides. Their application in quantitative immunofluorescence studies and tracing monoaminergic neuronal connections.

We have described immunocytochemical methods for the direct visualization of monoamine- and cGMP-containing neurons, using antibodies to the primary or secondary messengers themselves. The specificity and affinity of these antibodies were determined using quantitative immunofluorescence of gelatin models, as non-biological models, to which the native substances and their possibly cross-reacting compounds were incorporated. The use of retro- and anterograde tracers both in conjunction with immunohistochemical procedures provides the possibility to characterize transmitter specifically afferent and efferent pathways. With the presented double-label immunocytochemical procedures it is feasible to visualize and trace a projection between two brain areas, and in addition to specify the transmitters which are involved in their afferents and efferents and their target neurons and neurons of origin. Using the SCG, as a biological model, we could demonstrate that cGMP levels in individual cells could be elevated by cholinergic agonists. Moreover, using quantitative immunofluorescence of cGMP as a post- or presynaptic marker we have the possibility to measure the post-/presynaptic effects of monoamines or neuropeptides in individual neurons.

Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons.

By the neuroanatomical tracing technique based on uptake, transport, and immunocytochemical detection of injected Phaseolus vulgaris leucoagglutinin (PHA-L), fiber trajectories of labeled neurons can be followed with great accuracy to their termination areas. To further analyze the connectivity of these fibers, the target neurons must be chemically characterized. In vibratome and frozen sections of rat brain, we tried to visualize PHA-L-labeled fibers and, simultaneously, the target neuron-related antigen. As a model system we used the projection from the pre-frontal cortex to histaminergic neurons in the posterior hypothalamic region. We tested "sequential" and "pooled" immunocytochemical procedures. In the sequential procedure, the two antigens are detected by two successive and complete immunocytochemical staining procedures, with primary antibodies raised in different animal species and with different chromogens for the final visualization. In the pooled procedure, the sections are incubated with mixtures of primary and secondary antibodies, after which the procedure is similar to the sequential procedure. We obtained excellent results on vibratome sections with a sequential procedure using first conventional peroxidase immunocytochemistry (goat anti-PHA-L primary antibody) to visualize the transported PHA-L (brown reaction product), and subsequently alkaline phosphatase immunocytochemistry (rabbit anti-histidine decarboxylase primary antibody) to locate the histaminergic neurons (blue reaction product). The resulting preparations deteriorate, however, after 1-2 months of storage. Good results were also obtained with a double peroxidase procedure on frozen sections, using nickel-enhanced diaminobenzidine to visualize the PHA-L (dark blue reaction product), and diaminobenzidine (brown reaction product) to visualize the second antigen. The quality of these preparations is permanent.

Projection from the prefrontal cortex to histaminergic cell groups in the posterior hypothalamic region of the rat. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with immunocytochemistry of histidine decarboxylase.

We investigated the projection from the infralimbic division of the prefrontal cortex (area 25) to histaminergic neurons in the posterior hypothalamic area. Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected in the prefrontal cortex of rats. Frozen brain sections were subjected to combined PHA-L and histidine decarboxylase (HDC)-peroxidase immunocytochemistry, using nickel-enhanced diaminobenzidine (blue reaction product) to visualize the transported PHA-L, and diaminobenzidine (brown reaction product) to visualize simultaneously the HDC-containing neurons. PHA-L-labeled fibers could be seen coursing in the capsula interna, leaving the telencephalon via the anterior thalamic radiation and the medial forebrain bundle. In the lateral and posterior hypothalamic areas, PHA-L-labeled fibers leave the medial forebrain bundle and traverse the nuclei containing HDC-immunoreactive neurons. Varicosities on the PHA-L-labeled fibers, the majority of which occur en passant, could be observed in close association with the HDC-immunoreactive neurons. The results suggest that the hypothalamic histaminergic neurons receive afferent synaptic input from neurons of the infralimbic division of the prefrontal cortex.