Neuron-specific expression of high-molecular-weight clathrin light chain.

High-molecular-weight forms of clathrin light chains LCa and LCb contain inserted sequences and are expressed in brain tissue but have not been observed in peripheral tissues. Monoclonal antibodies specific for the high-molecular-weight form of LCb and all forms of LCa were used to analyze their expression in different species and different neuronal cell types. High-molecular-weight light chains were found in bovine, rat, mouse, chicken, and human brain, indicating a conserved pattern of expression. Neuron-specific expression of the high-molecular-weight light chains was suggested by analysis of human brain gray matter and white matter. The former contained a higher proportion of light chains with insertion sequences. Immunohistochemical analysis localized the high-molecular-weight form of LCb to synapses and neuronal perikarya, but not to glial cells. Immunofluorescent labeling of cultured chicken dorsal root ganglia confirmed expression in neurons but not Schwann cells. These results indicate that the high-molecular-weight forms of clathrin light chains are restricted in expression and found in neuronal cells.

Synaptic connections of neurones identified by Golgi impregnation: characterization by immunocytochemical, enzyme histochemical, and degeneration methods.

For more than a century the Golgi method has been providing structural information about the organization of neuronal networks. Recent developments allow the extension of the method to the electron microscopic analysis of the afferent and efferent synaptic connections of identified, Golgi-impregnated neurones. The introduction of degeneration, autoradiographic, enzyme histochemical, and immunocytochemical methods for the characterization of Golgi-impregnated neurones and their pre- and postsynaptic partners makes it possible to establish the origin and also the chemical composition of pre- and postsynaptic elements. Furthermore, for a direct correlation of structure and function the synaptic interconnections between physiologically characterized, intracellularly HRP-filled neurones and Golgi-impregnated cells can be studied. It is thought that most of the neuronal communication takes place at the synaptic junction. In the enterprise of unravelling the circuits underlying the synaptic interactions, the Golgi technique continues to be a powerful tool of analysis.

Quantitative immunohistochemistry of synaptophysin in human neocortex: an alternative method to estimate density of presynaptic terminals in paraffin sections.

Currently available specific synaptic markers have made it possible to estimate the synaptic density by immunochemical techniques. In the present study we labeled the neocortical presynaptic terminals in histological sections of human autopsy tissue with a monoclonal antibody against synaptophysin. The characteristic granular neuropil reaction was quantified by measuring the average optical density (OD) in the different layers of the parietal cortex with the aid of image analysis equipment. The raw neuropil OD was corrected by subtracting the OD of the white matter in the same section. Our study showed that consistent microdensitometric results can be obtained on 5-microns paraffin sections from specimens with less than 8 hr of post-mortem time before fixation, incubated with 5 micrograms/ml of anti-synaptophysin. The corrected OD measurements were slightly larger in neocortical layers II, III, and V than in layers I, IV, and VI, but the differences were not statistically significant. In area 17, layer IV was denser than the others. We conclude that with certain precautions this method can be used to measure relative amounts of synaptophysin-like immunoreactivity and to infer the density of presynaptic boutons in human situations and in animal models.

Localization of voltage-sensitive sodium channels on the extrasynaptic membrane surface of mouse skeletal muscle by autoradiography of scorpion toxin binding sites.

Voltage-dependent sodium channels (Na+ channels) were localized by autoradiography on mouse skeletal muscle using both light and electron microscopy. 125I-scorpion toxins (ScTx) of both the alpha and beta type were used as probes. The specificity of labelling was verified by competitive inhibition with unlabelled toxin and by inhibition of alpha ScTx labelling in depolarizing conditions. Under light microscopy, the labelling of the myocyte surface appeared randomly distributed with both the alpha and beta toxins. No difference in the labelling density obtained with beta ScTx was observed between a 2 mm central segment of the fibre containing the endplate and an adjacent segment not containing the endplate. At the endplate, however, the beta ScTx binding site density was about seven fold higher at the edge of the synaptic primary clefts. This density decreased with distance from the synaptic cleft reaching the extrasynaptic value at 30-40 microns. An analysis of myocyte labelling using electron microscopy provided evidence for a specific, but very low labelling of the myocyte interior which can be attributed to the T-tubules. These results confirm a relatively high density of Na+ channels in a perijunctional zone about 50 microns in width, which could ensure the initial spread of the surface depolarization with a high safety factor, and a homogeneous distribution over the remaining surface with a low density evaluated at 5-10 per microns2. However, the very low labelling of T-tubules could be attributed mainly to a low density of tubular Na+ channels.

The distribution of intracellular acetylcholine receptors and nuclei in avian slow muscle fibres during establishment of distributed synapses.

The distribution of intracellular acetylcholine receptor was studied by 125I-alpha-bungarotoxin autoradiography as a measure of the local acetylcholine receptor synthesis at junctional and extrajunctional sites in single fibres of the developing anterior latissimus dorsi muscle of the chicken. Large (longer than 2 microns) acetylcholine receptor clusters characteristic of synaptic contacts were localized by immunofluorescence with anti-acetylcholine receptor antibodies. The distance between acetylcholine receptor clusters at embryonic day 11 was 166 +/- 10.5 microns and this distance did not increase despite growth until after 4 days posthatch. The distance between acetylcholine receptor clusters subsequently increased proportionately with the increase in the length of fibres. Intracellular acetylcholine receptors were labelled with 125I-alpha-BGT after first blocking cell-surface acetylcholine receptor with unlabelled alpha-BGT, and treatment with saponin. Intracellular acetylcholine receptor represented about 5-15% of total cellular acetylcholine receptor. Cycloheximide experiments indicated that 80-90% of intracellular acetylcholine receptor examined represented newly synthesized acetylcholine receptor. The spatial distribution of this pool, studied by autoradiography, was determined in relation to the acetylcholine receptor clusters labelled with anti-acetylcholine receptor antibody. Between embryonic day 11 and posthatch day 14 there was a continual increase in intracellular acetylcholine receptor at both junctional and extrajunctional parts of the fibres, but with the greater increases occurring at the junctional regions. Peaks of intracellular acetylcholine receptor became associated with an increasing number of acetylcholine receptor clusters so that by posthatch day 14 there was an 80% correspondence. The accumulation of newly synthesized intracellular acetylcholine receptor under acetylcholine receptor clusters was not the result of the aggregation of nuclei at these sites, suggesting that a higher rate of acetylcholine receptor synthesis per nucleus develops at distributed synaptic sites on anterior latissimus dorsi fibres.

Monoamine synaptic structure and localization in the central nervous system.

The monoamines dopamine, noradrenaline, adrenaline, and serotonin as well as the diamine histamine have a widespread distribution in the central nervous system within synaptic terminals and nonsynaptic varicosities. In certain regions of the central nervous system the monoamines are contained in varicosities that have no synaptic specialization associated with them, suggesting a possible neuromodulatory role for some of the monoamines. The majority of monoamine labelled structures are synaptic terminals which are characterized by the presence of small, clear vesicles (40-60 nm) and large, granular vesicles (70-120 nm) within the terminal. A third population of vesicles--small, granular vesicles--which are visible only after histochemical staining, are probably the equivalent of the small, clear vesicles present after either autoradiographic or immunohistochemical labelling. Most monoamine containing terminals contact dendrites and dendritic spines and, less frequently, neuronal somata and other axons. Both asymmetrical and symmetrical membrane specializations are associated with monoaminergic terminals; however, asymmetrical contacts are the most frequent type found. These ultrastructural results indicate that monoamine containing terminals and varicosities in general share many common morphological features, but still have diverse functions.

GABAergic synapses in the brain identified with antisera to GABA and its synthesizing enzyme, glutamate decarboxylase.

GABA is a known inhibitory neurotransmitter in the mammalian brain. The site of GABAergic synapses can be determined with immunocytochemical methods that localize either GABA or its synthesizing enzyme, glutamate decarboxylase (GAD). In general, GABAergic axon terminals contain pleomorphic synaptic vesicles and form symmetric synapses. However, a small number of GABAergic axon terminals in selected brain regions (spinal cord, cerebellum, superior colliculus, striatum, globus pallidus, inferior olive, and substantia nigra) form asymmetric synapses. GAD- and GABA-immunoreactive processes that contain synaptic vesicles participate in every known morphological type of chemical synapse. These include axosomatic, axodendritic, axospinous, initial segment, axoaxonic, dendrodendritic, serial, reciprocal, and ribbon synapses. Although GABAergic synapses form a heterogeneous group, they most commonly form axosomatic, axodendritic, and initial segment synapses in the brain and spinal cord. These findings provide helpful guidelines for the identification of GABAergic synapses in future studies.

Ultrastructural description of glutamate-, aspartate-, taurine-, and glycine-like immunoreactive terminals from five rat brain regions.

The ultrastructural localization of putative excitatory (glutamate, aspartate) and inhibitory (taurine, glycine) amino acid neurotransmitters is described in several selected rat brain regions. In general, axon terminal profiles immunoreactive for excitatory amino acids formed asymmetric synapses with non-immunoreactive small diameter dendritic profiles or dendritic spines. In the cerebellum, both mossy fiber terminals and parallel fiber terminals were immunoreactive for glutamate and aspartate. In the hippocampus, mossy fiber terminals within the stratum lucidum of the CA3 region were immunoreactive for glutamate. Localization of glutamate and aspartate to cerebellar parallel and mossy fibers, as well as the identification of glutamate in hippocampal mossy fibers, is consistent with the excitatory nature of these fibers as described in previous physiological studies. Glutamate-like immunoreactive terminals were also identified in subnucleus caudalis of the spinal trigeminal nucleus and in the dorsal horn of the spinal cord. Immunoreactive axon terminals for two putative inhibitory neurotransmitters, glycine and taurine, displayed a greater number of morphological variations in synaptic structure. In the cerebellum, taurine-like immunoreactivity was present in both basket cell axon terminals which formed symmetric synapses with Purkinje cell neurons, and in a few mossy fiber terminals which formed asymmetric synapses with dendritic spines. In the area dentata of the hippocampus, taurine-like immunoreactive profiles formed asymmetric synapses with dendritic elements. Glycine-like immunoreactive terminals formed symmetric synapses with cell perikarya in both the ventral horn of the spinal cord and in the cochlear nuclei, and on axon terminals in the spinal trigeminal and cochlear nuclei. In contrast, some glycine-like immunoreactive terminals formed asymmetric synapses with distal dendritic profiles in the spinal cord and spinal trigeminal nucleus. The localization of taurine to cerebellar basket cell axons and glycine to axon terminals that synapse on ventral horn motor neuron perikarya is consistent with the hypothesis that these amino acids are functioning as inhibitory neurotransmitters at these synapses. Taurine localization to cerebellar mossy fibers and to fibers in the molecular layer of the dentate gyrus may be more consistent with a proposed neuromodulator role of taurine.

Glutamate-like immunoreactivity in the retina of a marine teleost, the dragonet.

The localisation of endogenous glutamate in the dragonet retina was investigated by light microscopic postembedding silver-enhanced immunogold labeling after incubation with an anti-glutamate antiserum. Rod and cone inner segments and synaptic terminals, as well as the inner plexiform layer, are moderately labeled. Bipolar cells and ganglion cell bodies show strong labeling. In the dorsal inner plexiform layer, the levels with square-patterned bipolar synaptic boutons can be identified by their prominent glutamate-immunoreactivity. These results support the idea that the majority of the neurons that constitute the direct, centripetal pathways through the retina use glutamate as their neurotransmitter.

Distribution of DARPP-32 in the basal ganglia: an electron microscopic study.

DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, has been studied by light and electron microscopical immunocytochemistry in the rat caudatoputamen, globus pallidus and substantia nigra. In the caudatoputamen, DARPP-32 was present in neurons of the medium-sized spiny type. Immunoreactivity for DARPP-32 was present in dendritic spines, dendrites, perikaryal cytoplasm, most but not all nuclei, axons and a small number of axon terminals. Immunoreactive axon terminals in the caudatoputamen formed symmetrical synapses with immunolabeled dendritic shafts or somata. Neurons having indented nuclei were never immunoreactive. In the globus pallidus and substantia nigra pars reticulata, DARPP-32 was present in myelinated and unmyelinated axons and in axon terminals. The labelled axon terminals in these regions formed symmetrical synaptic contacts on unlabelled dendritic shafts or on unlabelled somata. These data suggest that DARPP-32 is present in striatal neurons of the medium-sized spiny type and that these DARPP-32-immunoreactive neurons form symmetrical synapses on target neurons in the globus pallidus and substantia nigra. The presence of DARPP-32 in these striatal neurons and in their axon terminals suggests that DARPP-32 mediates part of the response of medium-size spiny neurons in the striatum to dopamine D-1 receptor activation.

Development of topographic connections between the isthmic nuclei and optic tecta in the frog Limnodynastes dorsalis.

In the frog Limnodynastes dorsalis, the pattern of topographic connections between the isthmic nuclei and optic tecta was determined by anterograde and retrograde transport of horseradish peroxidase from localised tectal regions. In both larvae and adults, reciprocal mapping of the uncrossed isthmo-tectal and tecto-isthmal projections was evidenced by the juxtaposition of labelled tecto-isthmal terminations with labelled cells in the cortex and medulla of the ipsilateral isthmic nucleus. The crossed isthmo-tectal projection was revealed by labelled cells in the cortex and medulla of the nucleus contralateral to the injection. In adults, rostral tectal areas projected to rostral and ventral regions of the ipsilateral isthmic nucleus. Following more caudal tectal injections, labelled cells were found in progressively more dorsal locations within the nucleus. Labelled cells in the contralateral nucleus were found in the rim cortex abutting a neuropil and in medullary cells adjacent to this region. Connections between ventral isthmic regions and most rostral tectum and between dorsomedial nucleus and caudomedial tectum were similar in both nuclei. However, for isthmic areas projecting to rostromedial and mid-tectum, the location of labelled cells in the contralateral nucleus was inverted with respect to the ipsilateral nucleus. This inversion would allow both nuclei to project to visually corresponding regions of each tectum. During larval stages the basic adult topography was established despite the continued neurogenesis of both isthmic nuclei and optic tecta. In late larval stages a rim neuropil appeared adjacent to the cortical region in the isthmic nuclei where labelled cells of the crossed isthmotectal projection were found. Prior to this stage labelled cells abutted labelled medullary cells. The appearance of this neuropil was approximately temporally correlated with the onset of electrophysiologically detectable responses in the ipsilateral visuotectal projection. Formation of the rim neuropil may relate to maturation of the tecto-isthmo-tectal connections which underlie this visual projection.

Synaptic organization of septal projections in the rat medial habenula: a wheat germ agglutinin-horseradish peroxidase and immunohistochemical study.

The synaptic organization of septal inputs to the rat habenular complex of the dorsal diencephalon was examined employing the anterograde tracer wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). The cellular distribution of substance P (SP) and choline acetyltransferase (ChAT) immunoreactivity was also studied at the light and electron microscopic level. Following placements of tracer within the entire septum, labeled axons were observed in the stria medullaris and in the medial and lateral subnuclei of the habenula. Following injections of tracer in the nuclei triangularis and septofimbrialis of the posterior septum, the medial subnucleus was heavily labeled, whereas the lateral subnucleus was devoid of peroxidase activity. The medial subnucleus possessed labeled myelinated axons and terminals that contained clear, spherical vesicles and formed asymmetric contacts with dendritic spines and shafts. Terminals possessing WGA-HRP activity also formed non-synaptic junctions with other labeled or unlabeled terminals. SP and ChAT immunoreactivity in normal and colchicine-treated animals was confined to dendrites and somata within the medial habenula. Terminals containing clear spherical vesicles formed asymmetric synaptic contacts with these immunoreactive somatic and dendritic profiles. Based on the combined anterograde tracing and immunohistochemical data, it is proposed that septal projections provide a direct innervation to habenular neurons that contain ChAT or SP activity. These septal inputs may play an important role in the facilitation of the ChAT- and SP-positive habenular neurons, both of which provide prominent afferent inputs to the interpeduncular nucleus. Thus, neurons of the habenula and interpeduncular nucleus are under the direct and indirect influence of septal neurons within the limbic forebrain circuit.

Ultrastructure of primary afferent fibres and terminals expressing alpha-galactose extended oligosaccharides in the spinal cord and brainstem of the rat.

The ultrastructural characteristics of primary afferent fibres, which express alpha-galactose extended oligosaccharides recognized by LD2 and LA4 monoclonal antibodies, and the subcellular localization of these oligosaccharides were studied. LD2 and LA4 antibodies both label intensely the plasma membrane of primary afferent fibres, and with LD2 antibody all immunoreactive profiles also possessed strong intracellular staining. In contrast, intracellular staining with LA4 antibody was observed in only a subpopulation of stained profiles. LD2-immunoreactive fibres were detected in trigeminal and Lissauer tracts and in lamina I (LI) and lamina II (LII), and appeared as a mixture of unmyelinated and myelinated fibres. The highest density of LD2-immunoreactive synaptic boutons was found in lamina II outer (LIIo). Many of the terminals were simple dome-shaped terminals, making single asymmetric synapses over small and medium-sized dendritic shafts and dendritic spines. All LA4-immunoreactive fibres were unmyelinated. In addition, some small scalloped central-glomerular terminals contacting two or three dendrites were found. LA4-immunoreactive fibres were found more frequently than terminals and appeared most heavily immunostained in trigeminal and Lissauer tracts. In the neuropil of LI and LII, LA4 profiles were generally very weakly immunostained, although a small sample of immunostained synaptic boutons was detected. All LA4-immunoreactive terminals were found in lamina II inner (LIIi) and made simple asymmetric axodendritic synapses. In addition to axons and terminals, some dendrites exhibited LD2 immunoreactivity and this was most intense in the region of synaptic vesicles. In addition to neurons, some endothelial cells were immunostained with LD2 antibody and astrocytes were immunostained with LA4 antibody.

Glutamate-like immunoreactivity in calyces of Held.

The pattern of glutamate-like immunoreactivity was investigated in the medial nucleus of the trapezoid body of adult rats. A monoclonal 'anti-glutamate' antibody was combined with postembedding immunohistochemistry involving a silver-intensified peroxidase-antiperoxidase procedure for light microscopy or an immunogold method for electron microscopy. The most conspicuous glutamate-immunoreactive elements were labelled calyces of Held. Because of the outstanding size of these terminals they may provide an attractive model for studying the details of glutamatergic neurotransmission at a synapse of the mammalian CNS.

Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat.

Thyrotropin-releasing hormone-like immunoreactivity (TRH-LI) was localized at the ultrastructural level in the dorsal vagal complex (DVC: dorsal motor nucleus of the vagus (DMV) and the nucleus of the solitary tract (NST] in rat. TRH-LI was concentrated in large granular vesicles in axons, presynaptic terminals, and non-synaptic axon varicosities. TRH-LI presynaptic terminals established both asymmetric and symmetric synaptic contacts with dendrites. These observations are consistent with recently described direct inhibitory and facilitatory effects of TRH on the electrical activity of neurons in the DVC.

Immunohistochemical quantification of the synapse-related protein synaptophysin in Alzheimer disease.

Alzheimer disease (AD) as well as other dementing disorders are characterized by a continuous loss of neurons in cortical and subcortical areas and probably by an extensive synaptic loss. In order to substantiate and localize the loss of synapses in AD, we quantified by microdensitometry the neuropil immunoreactivity to an antibody that labels the protein synaptophysin (p38), which is localized in the presynaptic terminals. We found in the AD cases an average 50% decrease in the density of the granular neuropil immunoreaction in parietal, temporal and midfrontal cortex. In contrast, Pick disease cases presented close to normal values in parietal cortex, but major losses in temporal and frontal cortex. Our data strongly suggest an important role of synapse loss in dementia.

Synaptic plasticity in the hypoglossal nucleus of female canaries: structural correlates of season, hemisphere, and testosterone treatment.

The caudal portion of the hypoglossal nucleus (nXIIts) contains the motor neurons that control the syrinx in songbirds. In canaries, song occurs seasonally, is principally produced by males, and appears to be produced predominantly by muscles on the left side of the syrinx. The present study measures the effect of seasonal change and manipulation of testosterone levels on synapse number and morphology in nXIIts in adult female canaries. We find that synapse density is lower in testosterone-treated birds than in control birds and lower in fall than in spring. The number of vesicles per presynaptic profile increases in the spring as a result of a general increase in this measure in all synapses. The number of vesicles per presynaptic profile also increases with testosterone treatment, primarily due to an increase in the proportion of synapses associated with unusually high vesicle counts. Together, these changes suggest that large reserves of neurotransmitter may be necessary to sustain singing. Several ultrastructural differences between hemispheres are found. Postsynaptic thickenings are longer, and postsynaptic processes are larger on the left side than on the right side. In the spring, there are more vesicles per synapse on the left than on the right, but this lateralization is reversed in the fall. Thus, lateralization of song production is associated with lateral asymmetries in synapse morphology. These hemispheric differences are relatively small, like those seen at the light microscope level, encouraging further consideration of peripheral as well as CNS sources of functional lateralization. The seasonal and testosterone-induced changes in synapse number and morphology may be components of the periodic reorganization of canary vocalization.

The connections from botzinger expiratory neurons to upper cervical inspiratory neurons in the cat.

These experiments examined possible inhibitory inputs to upper cervical inspiratory neurons from the expiratory neurons of the Botzinger complex. Eighty-one Botzinger neurons were tested with antidromic mapping for a projection to the C1 segment of the spinal cord; 44/81 (54%) were found to project, 27/79 (34%) contralaterally, 17/68 (25%) ipsilaterally, and 1/66 (2%) both contralaterally and ipsilaterally. Antidromic mapping in contralateral C1 demonstrated the presence of a collateral in 3/15 (20%) of the Botzinger neurons tested, while 3/9 (33%) had collateral arborizations in ipsilateral C1. The collaterals mapped were not localized to the region of the upper cervical inspiratory neurons. Microstimulation in C3 (12-17 microA, 0.2-ms duration) at locations which produced short-latency (2.7-3.5 ms) inhibition of phrenic nerve discharge resulted in the short latency (3.0 ms) inhibition of 1/27 (3.7%) upper cervical inspiratory neurons as demonstrated by cross-correlation. It was concluded that while some upper cervical inspiratory neurons may be inhibited during expiration by the Botzinger expiratory neurons, this connection is not a strong one.

High performance thin-layer chromatography and densitometry of synaptic plasma membrane lipids.

Previously, it has been shown that phospholipids, cholesterol, and glycolipids could be quantitated using the same high performance thin-layer chromatography (HPTLC) method. Here we examined that method in terms of linearity of standards in the nanogram range, recovery of nonacidic and acidic lipids after Sephadex column chromatography, and quantitation of lipids in mouse synaptic plasma membranes (SPM) where lipid content is low. Nonacidic and acidic fractions were separated by Sephadex column chromatography, applied to plates using contact spotting, chromatographed, visualized with cupric acetate, and quantitated using in situ densitometry. Recovery of nonacidic and acidic fractions off the columns was determined with radiolabeled phospholipids. Standards for each lipid class were linear in the nanogram range. Quantitation of SPM lipid classes could be made with as little as 1.5 micrograms of total lipid. Recovery of the nonacidic fraction after Sephadex column chromatography was approximately 100% whereas the acidic fraction was approximately 91%. Phospholipids, cholesterol, and glycolipids could be determined in nanogram amounts using the same method. This method is an efficient method for examining different lipid classes and in samples where lipid content is low.

Corticotropin-releasing factor synapses within the paraventricular nucleus of the hypothalamus.

Corticotropin-releasing factor (CRF) regulates the release of adrenocorticotropin (ACTH) from the anterior pituitary and these neurosecretory neurons reside in the paraventricular nucleus of the hypothalamus (PVN). In addition to its role as an ACTH secretogogue, exogenously administered CRF can act centrally to modify sympathetic outflow, alter various stress-induced behaviors and modulate its own secretion. Some of these effects might be mediated by CRF acting synaptically within the PVN as the nucleus is known to play a major role in integration of autonomic function. The current ultrastructural immunocytochemical study was designed to examine the range of synaptic relationships that CRF terminals make within the PVN. CRF-positive synapses were numerous, particularly in the periventricular zone. The majority of terminals formed axo-dendritic synapses and of these over 85% were the Gray's type II (symmetrical) class. Axo-somatic terminals were also encountered and both parvicellular and magnocellular neurons were innervated. Once again most of the terminals were Gray's type II. Although an innervation of unidentified structures was the most common, CRF synapses onto CRF neurons and dendrites were observed. All CRF/CRF interactions had symmetrical membrane specializations. These studies indicate that CRF could play a prominent role in the modulation of both parvicellular and magnocellular neurons within the paraventricular nucleus, including modulation of its own neurosecretory activity.