Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.

The cause of neurodegeneration in Huntington's disease (HD) is unknown. Patients with HD have an expanded NH2-terminal polyglutamine region in huntingtin. An NH2-terminal fragment of mutant huntingtin was localized to neuronal intranuclear inclusions (NIIs) and dystrophic neurites (DNs) in the HD cortex and striatum, which are affected in HD, and polyglutamine length influenced the extent of huntingtin accumulation in these structures. Ubiquitin was also found in NIIs and DNs, which suggests that abnormal huntingtin is targeted for proteolysis but is resistant to removal. The aggregation of mutant huntingtin may be part of the pathogenic mechanism in HD.

Compositional changes of AP-1 DNA-binding proteins are regulated by light in a mammalian circadian clock.

Recent reports have shown that the nuclear phosphoprotein Fos is induced by light in a mammalian circadian clock, the suprachiasmatic nucleus. To learn how light and circadian phase affect the binding of Fos to DNA, we analyzed the photic and temporal regulation of immunoreactive Jun protein expression and AP-1 DNA-binding activity in the rat suprachiasmatic nucleus. Immunohistochemistry and gel mobility shift assays suggest that AP-1 activity during the night and after a light pulse consists of constant, as well as variable, protein components; JunD could be identified as a constituent of both dark- and light-activated binding complexes, whereas binding by JunB and Fos could be implicated only after photic stimulation. Since JunD or JunB could be colocalized with Fos in individual suprachiasmatic nucleus cell nuclei, light may be acting in at least some suprachiasmatic nucleus cells by altering AP-1 protein composition rather than binding site occupancy.

CAG expansion affects the expression of mutant Huntingtin in the Huntington's disease brain.

A trinucleotide repeat (CAG) expansion in the huntingtin gene causes Huntington's disease (HD). In brain tissue from HD heterozygotes with adult onset and more clinically severe juvenile onset, where the largest expansions occur, a mutant protein of equivalent intensity to wild-type huntingtin was detected in cortical synaptosomes, indicating that a mutant species is synthesized and transported with the normal protein to nerve endings. The increased size of mutant huntingtin relative to the wild type was highly correlated with CAG repeat expansion, thereby linking an altered electrophoretic mobility of the mutant protein to its abnormal function. Mutant huntingtin appeared in gray and white matter with no difference in expression in affected regions. The mutant protein was broader than the wild type and in 6 of 11 juvenile cases resolved as a complex of bands, consistent with evidence at the DNA level for somatic mosaicism. Thus, HD pathogenesis results from a gain of function by an aberrant protein that is widely expressed in brain and is harmful only to some neurons.

Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy.

An expansion of polyglutamines in the N terminus of huntingtin causes Huntington's disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons. How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur. We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink. Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin. Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes, mitochondria, and nuclear membranes. Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes. Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles. We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.

Changes in cortical and striatal neurons predict behavioral and electrophysiological abnormalities in a transgenic murine model of Huntington's disease.

Neurons in Huntington's disease exhibit selective morphological and subcellular alterations in the striatum and cortex. The link between these neuronal changes and behavioral abnormalities is unclear. We investigated relationships between essential neuronal changes that predict motor impairment and possible involvement of the corticostriatal pathway in developing behavioral phenotypes. We therefore generated heterozygote mice expressing the N-terminal one-third of huntingtin with normal (CT18) or expanded (HD46, HD100) glutamine repeats. The HD mice exhibited motor deficits between 3 and 10 months. The age of onset depended on an expanded polyglutamine length; phenotype severity correlated with increasing age. Neuronal changes in the striatum (nuclear inclusions) preceded the onset of phenotype, whereas cortical changes, especially the accumulation of huntingtin in the nucleus and cytoplasm and the appearance of dysmorphic dendrites, predicted the onset and severity of behavioral deficits. Striatal neurons in the HD mice displayed altered responses to cortical stimulation and to activation by the excitotoxic agent NMDA. Application of NMDA increased intracellular Ca(2+) levels in HD100 neurons compared with wild-type neurons. Results suggest that motor deficits in Huntington's disease arise from cumulative morphological and physiological changes in neurons that impair corticostriatal circuitry.

Diminished flare response in neuropathic diabetic patients. Comparison of effects of substance P, histamine, and capsaicin.

The flare response in skin largely depends on an intact primary sensory fiber, the C-fiber. We measured the flare response to the intradermal injection of substance P, histamine, and capsaicin in control subjects and in diabetic patients with and without clinically obvious polyneuropathy. The neuropathic diabetic patients had a reduced flare response to substance P, histamine, and capsaicin, compared with control and nonneuropathic diabetic subjects. The smaller flare response in the neuropathic diabetics after capsaicin administration suggested a dysfunction of the peripheral component of the C-fiber. Alternatively, dysfunction of the mast cell or vascular reactivity may contribute to the diminished flare. Because C-fibers participate in nociception in addition to the flare response, the findings of this study, by a method that permits a quantifiable measurement of the function of peripheral sensory neurons in diabetic subjects, has potential usefulness in evaluating sensory neuropathy in diabetic patients.

Unexpected c-fos gene expression in the suprachiasmatic nucleus of mice entrained to a skeleton photoperiod.

Several authors have suggested that the transcriptional regulatory protein c-Fos might be part of the mechanism for photic entrainment of the circadian pacemaker in the suprachiasmatic nucleus (SCN) to environmental light:dark cycles. This hypothesis has been based on evidence gathered using single light pulses administered acutely to animals free-running in constant darkness. In order to begin to analyze SCN c-fos gene expression in animals during steady-state entrainment to photic cycles, we exposed male BALB/c mice to a skeleton photoperiod consisting of two 1-h light pulses separating a long (14 h) and a short (8 h) dark interval. The cycle was designed so that stable entrainment could be achieved in either one of two patterns (with rhythmic locomotor activity occurring during either the long or the short dark interval); SCN c-fos mRNA levels could then be measured during entrainment to light pulses at different phases of the circadian cycle, while controlling for the duration of preceding darkness. We found that c-fos was induced equally well by a light pulse that represented ZT 12 or ZT 3. The ZT 12 pulse functioned as an entraining pulse, because animals free-ran after it was removed from the lighting regimen, whereas removing the ZT 3 pulse caused little or no phase shift of activity onset. The data confirm that the expression of SCN c-Fos is not itself sufficient to reset rhythm phase, and they indicate that the role of this gene in the mechanism of photic entrainment is not yet fully understood.

Axonal transport of N-terminal huntingtin suggests early pathology of corticostriatal projections in Huntington disease.

Aggregation of N-terminal mutant huntingtin within nuclear inclusions and dystrophic neurites occurs in the cortex and striatum of Huntington disease (HD) patients and may be involved in neurodegeneration. We examined the prevalence of inclusions and dystrophic neurites in the cortex and striatum of 15 adult onset HD patients who had mild to severe striatal cell loss (grades 1, 2 or 3) using an antibody that detects the N-terminal region of huntingtin. Nuclear inclusions were more frequent in the cortex than the striatum and were sparse or absent in the striatum of patients with low-grade striatal pathology. Dystrophic neurites occurred in both regions. Patients with low-grade striatal pathology had numerous fibers with immunoreactive puncta and large swellings within the striatal neuropil, the subcortical white matter, and the internal and external capsules. In the globus pallidus of 3 grade 1 cases, N-terminal huntingtin markedly accumulated in the perinuclear cytoplasm and in some axons but not in the nucleus. Findings suggest that in the earlier stages of HD, accumulation of N-terminal mutant huntingtin occurs in the cytoplasm and is associated with degeneration of the corticostriatal pathway.

Early and progressive accumulation of reactive microglia in the Huntington disease brain.

Microglia may contribute to cell death in neurodegenerative diseases. We studied the activation of microglia in affected regions of Huntington disease (HD) brain by localizing thymosin beta-4 (Tbeta4), which is increased in reactive microglia. Activated microglia appeared in the neostriatum, cortex, and globus pallidus and the adjoining white matter of the HD brain, but not in control brain. In the striatum and cortex, reactive microglia occurred in all grades of pathology, accumulated with increasing grade, and grew in density in relation to degree of neuronal loss. The predominant morphology of activated microglia differed in the striatum and cortex. Processes of reactive microglia were conspicuous in low-grade HD, suggesting an early microglia response to changes in neuropil and axons and in the grade 2 and grade 3 cortex, were aligned with the apical dendrites of pyramidal neurons. Some reactive microglia contacted pyramidal neurons with huntingtin-positive nuclear inclusions. The early and proximate association of activated microglia with degenerating neurons in the HD brain implicates a role for activated microglia in HD pathogenesis.

Hypothyroidism increases substance P concentrations in the heterotopic anterior pituitary.

The regulatory effects of thyroid hormone on adenohypophysial substance P (SP) were studied in heterotopically implanted anterior pituitaries. Three or four anterior pituitaries from 21-day-old rat pups were implanted under the renal capsule in 175- to 200-g adult rats. The donor and recipient animals were sex matched. One week after implantation, animals were thyroidectomized or sham operated. A separate group of animals received daily T4 treatment (1.5 g/100 g, ip). After 2 weeks, the native and heterotopic pituitaries were assayed for SP, TSH, PRL, and LH. Thyroidectomy resulted in a 3- to 10-fold increase in the SP concentration in both the heterotopic and native pituitaries compared to euthyroid values. T4 treatment suppressed the SP levels in the heterotopic pituitaries of the thyroidectomized rats. In contrast to the reduction of TSH concentrations in native pituitaries in thyroidectomized animals vs. controls, TSH concentrations in the heterotopic pituitaries of thyroidectomized rats were approximately 10 times greater than those in euthyroid animals. PRL concentrations were unaffected by hypothyroidism in native and heterotopic pituitaries. Thyroidectomy resulted in a decrease in LH concentrations in the native anterior pituitary, without affecting LH concentrations in the implanted pituitary. These findings indicate that a direct link from the hypothalamus to the anterior pituitary is not required for the pituitary SP response to hypothyroidism.

The differential effects of thyroid and gonadal hormones on substance P content in the anterior pituitary of the prepubertal rat.

The effects of thyroid and gonadal status on the content of substance P in the anterior pituitary (AP-SP) were examined in prepubertal rats. A sex difference in AP-SP is evident by age 50 days [males, 287 +/- 35 fmol/mg protein (mean +/- SE); females, 103 +/- 17; P less than 0.05], and this difference becomes greater by 75 days (males, 543 +/- 54; females, 146 +/- 11.5; P less than 0.01). Hypothyroidism was induced in male and female pups by giving lactating dams 0.1% methimazole (wt/vol) in their drinking water after parturition. There was a marked and significant increase in AP-SP in 21-day-old hypothyroid compared to euthyroid control pups. Male pups were made thyrotoxic by daily treatment with T4 (10 micrograms/rat, sc) from age 8 to 15 days. AP-SP was 4 times lower in the thyrotoxic than in the euthyroid pups (P less than 0.001). Rats ovariectomized at age 22 days and killed on day 35 revealed no change in AP-SP, in contrast to the rise in AP-SP in the ovariectomized adult rat. Female pups were treated with dihydrotestosterone (DHT; 50 micrograms/day) or testosterone (50 micrograms/day) from age 8-20 days. Neither androgen induced a change in AP-SP. Female pups which received estradiol (E2; 0.5 micrograms/day) or testosterone (75 micrograms/day) from age 8-20 days also had no change in AP-SP. As opposed to the lack of effect of E2 and DHT on AP-SP in female rats younger than 22 days, E2 (1 microgram/100 g BW daily) caused a decrease and DHT (100 micrograms/100 g BW daily) caused an increase in AP-SP in female rats treated from 22-35 days of age [E2, 91 +/- 6.9; DHT, 226 +/- 31 (P less than 0.05 vs. control for both); control, 154 +/- 13]. We conclude that the responsiveness of AP-SP to alterations in thyroid status is present at the youngest age studied. In contrast, the responsiveness of AP-SP to changes in the levels of gonadal steroids is absent in the infantile period and requires a maturational process that becomes evident during the juvenile state of sexual development.

Regulation by thyroid hormone of the concentration of substance P in the rat anterior pituitary.

The content of immunoreactive substance P (iSP) in the male rat anterior pituitary was measured after thyroidectomy and excess T4 administration. Baseline values for iSP content (mean +/- SE, 400 +/- 37 fmol/mg protein) increased progressively after thyroidectomy (4 days, 893 +/- 100; 6 days, 1321 +/- 242; 14 days, 1897 +/- 509). Administration of pharmacological doses of T4 (50 micrograms daily) for 2 and 14 days significantly decreased anterior pituitary iSP content (2 days, 196 +/- 30; 14 days 138 +/- 12). Thyroid status did not affect iSP content in the hypothalamus, caudate nucleus, amygdala, or brainstem. Partial chemical characterization of SP immunoreactivity in the anterior pituitary was obtained by gel permeation chromatography on Sephadex G-25, high pressure liquid chromatography, and the use of two antisera in RIAs, one directed against the amino-terminus and one directed against the carboxyl-terminus of the peptide. SP in the anterior pituitary was readily releasable in vitro by 44 mM potassium chloride in a calcium-dependent manner. The present study demonstrates that the concentration of iSP in the rat anterior pituitary is affected by the thyroid status of the animal and supports the probability that thyroid hormones participate in the regulation of the synthesis and/or release of iSP from the anterior pituitary.

The effects of gonadal steroids on the content of substance P in the rat anterior pituitary.

The effects of gonadectomy and of the administration of gonadal steroids on the content of substance P in the anterior pituitary (AP-SP) were studied in adult rats. The effect of gonadal status on the AP-SP content of thyroidectomized (TX) rats was also studied. We have confirmed that the AP-SP content in adult males is higher than that in adult females. Ovariectomy (OVX) caused an increase in AP-SP content which was apparent 6 days after surgery. Estradiol (E2; 2 micrograms/rat daily) administered for 13 days beginning the day after OVX prevented the increase in AP-SP content induced by OVX. Orchiectomy of adult rats had no effect on AP-SP content 14 and 45 days after surgery. E2 administered to adult female rats for 13 days caused a reduction in the AP-SP content, whereas dihydrotestosterone (0.2 mg/rat daily for 13 days) caused an increase that was even more pronounced in TX rats. E2 administration to TX adult female rats caused a significant decrease in the AP-SP content both when treatment was begun on the day after surgery or 2 weeks later. Administration of T4 (1.5 and 25 micrograms/100 g BW daily for 7 days) to rats made hypothyroid by thyroidectomy 2 weeks earlier abolished the increase in AP-SP content seen in TX animals. Neither E2 nor dihydrotestosterone had an effect on the substance P content of any of the brain regions examined. The AP-SP content of pregnant or lactating rats was not different from that of age-matched controls. The content of substance P in the AP and median eminence did not vary significantly throughout the estrous cycle. The data indicate that AP-SP content is dependent on the gonadal status of the animal and that gonadal steroids interact with thyroid hormones in the regulation of substance P turnover in the AP.

Metastatic papillary thyroid carcinoma to lung diagnosed by bronchoalveolar lavage.

The diagnosis of papillary carcinoma of the thyroid metastatic to the lung frequently requires a battery of noninvasive tests. Occasionally, invasive procedures such as open lung biopsy, transthoracic needle biopsy, and transbronchial lung biopsy are employed to confirm the diagnosis. A 31-yr-old woman with papillary thyroid carcinoma treated previously by a near-total thyroidectomy and 131I ablation presented to our clinic with shortness of breath and a clear chest roentgenogram. A post-131I treatment whole body scan revealed widespread 131I pulmonary uptake, and the presence of papillary thyroid cancer was confirmed by bronchoalveolar lavage. We conclude that bronchoalveolar lavage should be considered when tissue confirmation of metastatic papillary carcinoma to the lung is needed. During the evaluation and follow-up of this patient, we were able to determine that metastatic papillary carcinoma to the lung may cause a methacholine bronchoprovocation test to be falsely positive for asthma.

Failure of bromocriptine to lower plasma catecholamines in normal men and women.

A plasma catecholamine-lowering effect of bromocriptine has previously been reported in normals, but not in patients with PRL- or GH-secreting pituitary tumors, and has been used as evidence to support the concept of disordered central nervous system dopaminergic tone in patients with such tumors and as a test to distinguish them from normals. In the present study of 16 normal subjects (9 women and 7 men), we found no significant change in plasma norepinephrine or epinephrine after bromocriptine. Similarly, no change occurred in plasma catecholamines in 8 patients with PRL-secreting tumors or 6 patients with ACTH hypersecretion. Our data, therefore, do not provide confirmatory evidence for an effect of bromocriptine on plasma catecholamines in normal subjects and do not support the proposed use of bromocriptine as a test for defective central dopaminergic regulation.

Impaired clearance of beta-lipotropin in uremia.

Immunoreactive ACTH and beta-lipotropin (beta-LPH) plasma concentrations are elevated in clinically stable chronic renal failure patients on hemodialysis (LPH: patients, 271.8 +/- 35.7 fmol ml-1; normal subjects; 6.6 +/- 0.5; ACTH: patients, 56.4 +/- 15.3; normal subjects, 19.4 +/- 1.7). To begin to study the etiology of such elevated levels, the MCR, apparent volume of distribution, and fractional rate of disappearance of synthetic human ACTH and highly purified human beta-LPH were determined in two clinically stable chronic renal failure patients on hemodialysis, after bolus simultaneous injection of both peptides. Biphasic disappearance curves were obtained for beta-LPH; triphasic for ACTH. The MCR of ACTH was within the range seen in normal subjects, whereas the MCR of beta-LPH was less than one half the normal rate. The data indicate that a decrease in MCR (rather than an increase in pituitary secretory rate) may account for the higher plasma levels of beta-LPH in uremic patients.

A kindred with a variant of multiple endocrine neoplasia type 1 demonstrating frequent expression of pituitary tumors but not linked to the multiple endocrine neoplasia type 1 locus at chromosome region 11q13.

Acromegaly is uncommon in kindreds with multiple endocrine neoplasia type 1 (MEN1), whereas primary hyperparathyroidism (PHP) has the highest penetrance of any endocrinopathy. We report an unusual MEN1 kindred with frequent expression of pituitary tumors and a low penetrance of PHP. Four members were found to have disease: PHP in generation I, acromegaly (2 cases) in generation II, and hyperprolactinemia associated with a pituitary tumor in generation III. There was no evidence for PHP in 1 patient with acromegaly (age 60 yr), the patient with hyperprolactinemia and the pituitary tumor (age 22 yr), and 1 asymptomatic obligate carrier (age 50 yr). Screening of 26 members revealed the possible diagnosis of PHP in 1 family member in generation II and possible early acromegaly in 2 members of generation III with elevated serum concentrations of insulin-like growth factor I and insulin-like growth factor-binding protein-3 but normal patterns of pulsatile GH release. Although the predisposing genetic defect in typical MEN1 families has previously been mapped to chromosome location 11q13 without evidence of heterogeneity among the 87 families analyzed, linkage of disease in this family to the MEN1 region is unlikely based on haplotype analysis. Localization of the gene(s) responsible for disease in such atypical families may aid in the understanding of the pathogenesis of MEN1. In addition, further study of the earliest changes in patterns of pulsatile GH release in familial acromegaly may allow more insight into the pathogenesis and natural history of this disease.

Immunoreactive leu-enkephalin in the monkey hypothalamus including observations on its ultrastructural localization in the paraventricular nucleus.

The distribution of immunoreactive leu-enkephalin neurons and fibers in the monkey hypothalamus, including ultrastructural localization in the paraventricular nucleus (PVN), was examined with the peroxidase-antiperoxidase immunocytochemical method. Immunoreactive leu-enkephalin cell bodies and fibers were present in the PVN, the region of the dorsal nucleus and nucleus of the anterior commissure, the dorsomedial nucleus, ventromedial nucleus, and lateral hypothalamus. Within the PVN labeled cells were found mostly in the medial parvocellular region, and a smaller proportion including some large cells was present in the lateral, and dorsolateral zones. Immunoreactive neurons contained numerous large granular vesicles (LGV) which ranged from 63 to 235 nm in size, suggesting that at least some enkephalin-containing neurons belong to the population of neurosecretory cells. Positive neurons were postsynaptic to four types of unlabeled axon terminals. Leu-enkephalin-containing fibers (some of which were myelinated) and boutons contained small clear vesicles and numerous LGV. Axon terminals made synaptic contacts with the cell bodies, primary and distal dendrites of unlabeled neurons. The findings show that enkephalin-containing neurons in the PVN integrate a variety of neuronal inputs and provide morphological evidence for the inhibiting influence of enkephalins on the firing rate of PVN neurons. It may be speculated that the effects of opioids on the release of vasopressin and other substances possibly originating from PVN neurons may be regulated in part within the nucleus by locally synapsing axons belonging to enkephalin-containing neurons.

Ultrastructural localization of immunoreactive calbindin-D28k in the rat and monkey basal ganglia, including subcellular distribution with colloidal gold labeling.

Normal cellular function depends on the controlled flux of Ca++ within intracellular compartments and across the plasma membrane. Proteins that bind Ca++ are thought to contribute to the regulation of intracellular Ca++ and, perhaps more importantly, signal functional changes in cell activity. In the brain, calbindin-D28k is among a class of calcium-binding proteins that are widely and heterogeneously distributed in select populations of neurons, among them neostriatal cells, but whose function is largely unknown. In this study of the monkey and rat neostriatum and globus pallidus, calbindin-D28k was localized with immunoperoxidase and immunogold methods in order to identify striatal cell populations that contain this protein and the subcellular compartments in which it is likely to function. Light and electron microscopy showed intense and extensive labeling of immunoreactive calbindin-D28k in the cell bodies, dendrites, and spines of medium-sized neostriatal spiny neurons and in their axon terminals which end in the globus pallidus. More discrete labeling with a gold-conjugated second antibody showed that the predominant site of calbindin-D28k was the matrix of the cytoplasm. Gold label was also associated with the karyoplasm of spiny cells and with the neurofilaments and axoplasmic matrix of striatopallidal axons and terminals, respectively. Membranes were either sparsely labeled (endoplasmic reticulum, mitochondria) or devoid of gold particles (nuclear envelope and plasmalemma). Radioimmunoassays of striatal subcellular fractions supported the anatomical findings by indicating that the soluble fractions of neostriatal tissue homogenates contained most of the calbindin-D28k immunoreactivity and that washes from forebrain synaptosomes treated with Triton X-100 yielded high levels of immunoreactive calbindin-D28k. These findings show that immunoreactive calbindin-D28k is localized to spiny neurons of the striatopallidal pathway and are consistent with previous observations on subcellular localization in nonneuronal tissues. If, as recently speculated, calbindin-D28k regulates calcium concentrations in neostriatal spiny neurons, this feature may be particularly involved with the high density of glutamatergic inputs to these cells. More work is needed to determine whether calbindin-D28k, when complexed to Ca++ in neostriatal spiny cells, signals the activation of protein kinases, phosphorylation, and/or neurotransmitter release, as has been shown for other Ca++-binding proteins in mammalian tissues.

A Golgi study of the monkey paraventricular nucleus: neuronal types, afferent and efferent fibers.

The neuronal organization of the paraventricular nucleus (PVN) was examined in Golgi impregnations of adult monkey. Results showed that at least six types of neurons could be identified in the nucleus on the basis of morphological features of the somata, dendrites, and axons. Four types of neurons with sparse to densely spined cell bodies and dendrites exhibited long axons and included large neurons (types I and II), medium-sized to large neurons (type III), and small to medium-sized cells (type IV). Axons of type I, III, and IV neurons had different diameters and were followed out of the PVN. Axon collaterals that arborized within the PVN were seen on the axons of types III and IV cells. Two types of interneurons with small somata were also found. One (type V) exhibited varicose dendrites and a profusely arborizing local axon. The other cell (type VI) had recurved dendrites with long appendages and no impregnated axon. Afferent fibers were also identified. Type 1 was a fine-caliber axon that coursed long distances in the PVN and exhibited numerous short branches. Additional observations suggested that type 1 afferents originated from the stria terminalis. The other afferent axon (type 2) was thicker and gave rise to terminal arborizations containing clusters of small swellings. The efferent fibers of the PVN were also examined in impregnations of the paraventriculosupraopticohypophysial tract. Fibers formed an extensive plexus as they coursed ventrally and passed through the lateral hypothalamus. Axons coursing more laterally in the tract were much larger than those more medially located. Our findings show a diverse organization of neuronal types within the monkey PVN with evidence for intrinsic connections through axon collaterals of efferent neurons and the locally arborizing axons of interneurons. Correlations are proposed between morphological subtypes of neurons seen in this Golgi study and the known functional output pathways of the PVN.