Immunocytochemical colocalization of progestin receptors and beta-endorphin or enkephalin in the hypothalamus of female guinea pigs

Double-label immunocytochemistry was used to determine whether estradiol-induced progestin receptors and either beta-endorphin or leucine-enkephalin are colocalized in female guinea pig brain. Ovariectomized, adult guinea pigs were implanted with capsules containing estradiol-17 beta to induce high levels of progestin receptors, and injected intracerebroventricularly with colchicine to improve visualization of the opiate peptides. Sections through the hypothalamus and preoptic area were processed for progestin receptor, followed by beta-endorphin or leucine-enkephalin immunocytochemistry. As reported previously, high concentrations of progestin receptor-immunoreactive (PR-IR) cells were found in the preoptic area (medial and periventricular portions, medial preoptic nucleus) and hypothalamus (anterior hypothalamic and arcuate nuclei, ventrolateral area). Many beta-endorphin-IR cells contained PR-IR in the arcuate nucleus and its surroundings (33%) and in the dorsomedial area of the hypothalamus (64%). Scattered enkephalin-IR cells were found in the septal nucleus, medial and lateral preoptic area, bed nucleus of the stria terminalis, and the arcuate nucleus. The ventromedial nucleus of the hypothalamus and dorsolateral magnocellular nucleus, respectively, contained moderate and heavy concentrations of enkephalin-IR cells. Although some of these areas also contained PR-IR, enkephalin-IR was colocalized consistently with PR-IR only in a small number of cells in the arcuate nucleus and ventromedial/ventrolateral area of the hypothalamus. These data, taken together with earlier observations that virtually all cells containing estradiol-induced PR-IR also contain estrogen receptor-IR, provide neuroanatomical evidence that hypothalamic actions of progesterone and estradiol may be mediated by beta-endorphin and/or enkephalin.     

Distribution of methionine- and leucine-enkephalin within the rat pituitary gland measured by highly specific radioimmunoassays

The distribution of methionine- and leucine-enkephalin within the rat pituitary gland was measured using highly specific antisera in conjunction with purification by high performance liquid chromatography. The highest concentrations were found in the pars intermedia (7 pmole/mg for methionine-enkephalin, 4 pmole/mg for leucine-enkephalin), whilst the pars nervosa contained 2.2 pmole/mg of each and the pars anterior the least (methionine-enkephalin: 0.51 pmole/mg, leucine-enkephalin: 0.36 pmole/mg).     

Thiamine-like fibers in the monkey brain: an immunocytochemical study

The distribution of thiamine-immunoreactive structures was studied in the brain of the monkey using an indirect immunoperoxidase technique. Fibers containing thiamine, but no thiamine-immunoreactive cell bodies, were found. The highest density of fibers containing thiamine was observed in the pulvinar nucleus and in the region extending from the pulvinar nucleus to the caudate nucleus. In the mesencephalon, immunoreactive fibers containing thiamine were only found at rostral level close to the medial lemniscus (at the mesencephalic-diencephalic junction). In the thalamus, the distribution of thiamine-immunoreactive structures was more widespread. Thus, immunoreactive fibers were found in nuclei close to the midline (centrum medianum/parafascicular complex), in the ventrolateral thalamus (medial geniculate nucleus, inferior pulvinar nucleus), and in the dorsolateral thalamus (lateral posterior nucleus, pulvinar nucleus). Finally, in the anterior commissure and in the cerebral cortex a low density immunoreactive fibers was visualized. Thus, in the brainstem, no immunoreactive structures were visualized in the medulla oblongata, pons, or in the medial-caudal mesencephalon, and no immunoreactive fibers were observed in the cerebellum, hypothalamus and in the basal ganglia. The present report describes the first visualization and the morphological characteristics (thick, smooth and short, medium or long in length) of the thiamine-immunoreactive fibers in the primate central nervous system using an antiserum directed against this vitamin. The distribution of thiamine-immunoreactive structures in the monkey brain suggests that this vitamin could be involved in several physiological mechanisms.     

Immunocytochemical colocalization of progestin receptors and β-endorphin or enkephalin in the hypothalamus of female guinea pigs

Double-label immunocytochemistry was used to determine whether estradiol-induced progestin receptors and either β-endorphin or leucine-enkephalin are colocalized in female guinea pig brain. Ovariectomized, adult guinea pigs were implanted with capsules containing estradiol-17β to induce high levels of progestin receptors, and injected intracerebroventricularly with co chicine to improve visualization of the opiate peptides. Sections through the hypothalamus and preoptic area were processed for progestin receptor, followed by β-endorphin or leucine-enkephalin immunocytochemistry. As reported previously, high concentrations of progestin receptor-immunoreactive (PR-IR) cells were found in the preoptic area (medial and periventricular portions, medial preoptic nucleus) and hypothalamus (anterior hypothalamic and arcuate nuclei, ventrolateral area). Many β-endorphin-IR cells contained PR-IR in the arcuate nucleus and its surroundings (33%) and in the dorsomedial area of the hypothalamus (64%). Scattered enkephalin-IR cells were found in the septal nucleus, medial and lateral preoptic area, bed nucleus of the stria terminalis, and the arcuate nucleus. The ventromedial nucleus of the hypothalamus and dorsolateral magnocellular nucleus, respectively, contained moderate and heavy concentrations of enkephalin-IR cells. Although some of these areas also contained PR-IR, enkephalin-IR was colocalized consistently with PR-IR only in a small number of cells in the arcuate nucleus and ventromedial/ventrolateral area of the hypothalamus. These data, taken together with earlier observations that virtually all cells containing estradiol-induced PR-IR also contain estrogen receptor-IR, provide neuroanatomical evidence that hypothalamic actions of progesterone and estradiol may be mediated by β-endorphin and/or enkephalin.     

Immunohistochemical localisation of natriuretic peptides in the heart and brain of the gulf toadfish Opsanus beta

Summary The distribution of natriuretic peptide immunoreactivity was determined in the heart and brain of the gulf toadfish Opsanus beta using the avidin-biotin peroxidase technique. Four antisera were used: the first raised against porcine brain natriuretic peptide which cross-reacts with atrial natriuretic and C-type natriuretic peptides (termed natriuretic peptide-like immunoreactivity); the second raised against porcine brain natriuretic peptide which cross-reacts with C-type natriuretic peptide but not with atrial natriuretic peptide (termed porcine brain natriuretic peptide-like immunoreactivity); the third raised against rat atrial natriuretic peptide; and the fourth raised against eel atrial natriuretic peptide. Natriuretic peptide- and porcine brain natriuretic peptide-like immunoreactivity was observed in all cardiac muscle cells of the atrium. In the ventricle, natriuretic peptide-like immunoreactivity was found in all cardiac muscle cells, however porcine brain natriuretic peptidelike immunoreactivity was confined to muscle cells adjacent to the epicardium. There was no discernible difference in the distribution of natriuretic peptide-like immunoreactivity and porcine brain natriuretic peptide-like immunoreactivity in the brain. Immunoreactive perikarya were observed only in the preoptic region of the diencephalon, and many immunoreactive fibres were found in the telencephalon, preoptic area, and rostral hypothalamus, lateral to the thalamic region. There was no immunoreactivity in any region of the hypophysis. A pair of distinct immunoreactive fibre tracts ran caudally from the preoptic area to the thalamic region, from which fibres extended to the posterior commissure, area praetectalis, dorsolateral regions of the midbrain tegmentum, and tectum. Many immunoreactive fibres were present in the rostral regions of the inferior lobes of the hypothalamus and in the dorsolateral and ventrolateral aspects of the rhombencephalon. No immunoreactivity was observed in the heart and brain using rat atrial natriuretic and eel natriuretic peptide antisera. Although the chemical structure of natriuretic peptides in the heart and brain of toadfish is unknown, these observations show that a component of the natriuretic peptide complement is similar to porcine brain natriuretic and/or porcine C-type natriuretic peptides. The presence of natriuretic peptides in the brain suggests that they could be important neuromodulators and/or neurotransmitters.     

Cholecystokinin octapeptide immunoreactivity in the nucleus tractus solitarius of the cat

The nucleus tractus solitarius possessed distinct patterns of cholecystokinin immunoreactive fibers and cell bodies within its various subdivisions. The commissural, medial, intermediate, parvocellular, dorsolateral and interstitial subdivisions contained relatively dense amounts of CCK immunolabelled fibers. In contrast, CCK immunoreactivity within the ventrolateral subdivision consisted of a few scattered fibers and small neurons. The commissural, intermediate, medial, dorsolateral and parvocellular subdivisions contained CCK immunoreactive neurons following colchicine treatment. The presence of CCK in the NTS suggest that it may be involved as a neuromodulator and/or neurotransmitter in circuitry that mediate cardiovascular, respiratory, gastrointestinal and taste functions.     

Histamine-immunoreactive neurons in the hypothalamus of cats

The localization of histaminergic neurons in the cat brain was determined immunohistochemically with an antibody against histamine. We found that histamine-immunoreactive neurons are observed exclusively in the posterior hypothalamus of colchicine treated cats. The larger group of neurons was found in the ventrolateral part of the posterior hypothalamus, including the tuberomammillary nucleus. Histamine-positive neurons were also observed in the supramammillary area and adjacent posterior hypothalamic area, as well as in the peri- and premammillary regions. In addition, numerous histamine immunoreactive fibers were detected, not only in the posterior hypothalamus, but also in other brain areas, such as the preoptic area of the anterior hypothalamus.     

Peptide YY-like immunoreactivity in the central nervous system of the rat

The concentration of peptide YY (PYY)-like immunoreactivity in rat brain and spinal cord was determined by radioimmunoassay. The highest concentrations were found in the cervical spinal cord (18.1 +/- 1.3 ng/g, mean +/- S.E.M.) and in the medulla oblongata (16.3 +/- 1.5 ng/g). Lower amounts were found in the pons and in the hypothalamus. Chromatographic analysis of the PYY-like immunoreactivity from various regions of the brain revealed 95% of the immunoreactive material to be indistinguishable from synthetic porcine PYY. PYY-immunoreactive nerve cell bodies could be demonstrated by immunocytochemistry in the medulla oblongata of colchicine-treated rats, the largest group of cells being found in the midline area between and partly in the raphe pontis and obscurus nuclei. Another large group of immunoreactive cells was detected more laterally in the medial parts of the gigantocellular reticular nucleus. A few cells, finally, were seen in the dorsal parts of the medulla, including the nucleus of the solitary tract. Varicose nerve fibers displaying PYY immunoreactivity were observed in many parts of the hypothalamus, pons, medulla and spinal cord.     

Effect of dorsolateral pontine lesions on diaphragmatic activity during REMS

Muscle atonia is a feature of normal rapid-eye-movement sleep (REMS). The suppression of accessory respiratory muscle activity has been investigated and a role for sleep-disordered breathing hypothesized, but the suppression of diaphragmatic activity has rarely been considered. We hypothesized that the activity of the diaphragm was suppressed by an area of the dorsolateral pons during REMS. Lesions in this region have previously been shown to abolish the atonia of REMS. The diaphragmatic electromyogram (EMG) activity was analyzed in five naturally sleeping cats before and after pontine lesions leading to REMS without atonia. Although respiratory timing parameters were not altered by the lesion, the inspiratory rate of rise was significantly increased in all cats, and the brief pauses (40-100 ms) in the diaphragmatic EMG normally seen in REMS were virtually abolished. We conclude that the dorsolateral pons has a role in suppressing diaphragmatic activation during REMS. This suppression affects the average rate of rise of diaphragmatic activity and also leads to brief intermittent complete cessation of ongoing muscle activity. These decrements in diaphragm activity could jeopardize ventilation during REMS.     

Immunocytochemical localization of corticotropin-releasing factor (CRF) in the rat brain

The immunocytochemical localization of neurons containing the 41 amino acid peptide corticotropin-releasing factor (CRF) in the rat brain is described. The detection of CRF-like immunoreactivity in neurons was facilitated by colchicine pretreatment of the rats and by silver intensification of the diaminobenzidine end-product. The presence of immunoreactive CRF in perikarya, neuronal processes, and terminals in all major subdivisions of the rat brain is demonstrated. Aggregates of CRF-immunoreactive perikarya are found in the paraventricular, supraoptic, medial and periventricular preoptic, and premammillary nuclei of the hypothalamus, the bed nuclei of the stria terminalis and of the anterior commissure, the medial septal nucleus, the nucleus accumbens, the central amygdaloid nucleus, the olfactory bulb, the locus ceruleus, the parabrachial nucleus, the superior and inferior colliculus, and the medial vestibular nucleus. A few scattered perikarya with CRF-like immunoreactivity are present along the paraventriculo-infundibular pathway, in the anterior hypothalamus, the cerebral cortex, the hippocampus, and the periaqueductal gray of the mesencephalon and pons. Processes with CRF-like immunoreactivity are present in all of the above areas as well as in the cerebellum. The densest accumulation of CRF-immunoreactive terminals is seen in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The widespread but selective distribution of neurons containing CRF-like immunoreactivity supports the neuroendocrine role of this peptide and suggests that CRF, similarly to other neuropeptides, may also function as a neuromodulator throughout the brain.     

Human brain natriuretic peptide-like immunoreactivity in human brain.

The presence of immunoreactive human brain natriuretic peptide in the human brain was studied with a specific radioimmunoassay for human brain natriuretic peptide-32. This assay showed no significant cross-reaction with human alpha atrial natriuretic peptide, porcine brain natriuretic peptide or rat brain natriuretic peptide. Immunoreactive human brain natriuretic peptide was found in all 5 regions of human brain examined (cerebral cortex, thalamus, cerebellum, pons and hypothalamus) (0.6-6.7 pmol/g wet weight, n = 3). These values were comparable to the concentrations of immunoreactive alpha atrial natriuretic peptide in human brain (0.5-10.1 pmol/g wet weight). However, Sephadex G-50 column chromatography showed that the immunoreactive human brain natriuretic peptide in the human brain eluted earlier than synthetic human brain natriuretic peptide-32. These findings suggest that human brain natriuretic peptide is present in the human brain mainly as larger molecular weight forms.     

Vasoactive intestinal polypeptide (VIP)-containing neurons: distribution throughout the brain of the chick (Gallus domesticus) with focus upon the lateral septal organ

The distribution of VIP-like perikarya and fibers was determined throughout the chick brain. The most rostral immunoreactive perikarya were found to be cerebrospinal fluid-contacting neurons in the pars medialis of the lateral septal organ. Additional data were presented supporting the idea that the lateral septal organ is another circumventricular organ within the brain of birds (Kuenzel and van Tienhoven 1982). A large group of immunoreactive perikarya was found in the lateral hypothalamic area and appeared continuous with immunoreactive neurons in the anterior medial and ventromedial hypothalamic nuclei (n). A few perikarya were located in the paraventricular hypothalamic n. A number of immunoreactive neurons were found within and about the infundibular and inferior hypothalamic n., none however was immunoreactive cerebrospinal fluid-contacting neurons. Immunoreactive perikarya were found predominantly in laminae 10–11 of the stratum griseum et fibrosum superficiale. A few scattered perikarya were found ventromedial to the n. tegmenti pedunculo-pontinus pars compacta and locus ceruleus. Some of the immunoreactivity was unusual, being very homogeneous within the cell body with little evidence of the material in the axon or dendrites. Perikarya were found in the central gray, n. intercollicularis, and area ventralis of Tsai. The most caudal structure showing immunoreactive neurons was the n. reticularis paragigantocellularis lateralis. Brain areas containing the most abundant immunoreactive fibers, listed from the rostral-most location, were found in the ventromedial region of the lobus parolfactorius and the lateral septal n. Continuing caudally, there were immunoreactive fibers within the periventricular hypothalamic n.; some of the fibers were found to travel for some distance parallel to the third ventricle. Dense immunoreactive fibers were found in the tractus cortico-habenularis et cortico-septalis, medial habenular n. and posterior and dorsal n. of the archistriatum. A number of areas had what appeared to be baskets of immunoreactive fibers (perhaps immunoreactive terminals) surrounding non-reactive perikarya. Brain areas containing terminals included the piriform cortex, area ventralis of Tsai, interpeduncular n., and specific regions of the stratum griseum et fibrosum superficiale. A very dense immunoreactivity occurred within the external zone of the median eminence, the dorsolateral parabrachial n., and n. tractus solitarii. Vasoactive intestinal polypeptide appears to be a useful peptide for defining the neuroanatomical constituents of the visceral forebrain in birds.     

Microtubules and protein secretion in rat lacrimal glands: localization of short-term effects of colchicine on the secretory process

The pathway and kinetics of the secretory protein transport in rat lacrimal exorbital gland have been established by an in vitro time- course radioautographic study of pulse-labeled protein secretion. The colchicine-sensitive steps have been localized by using the drug at various times with respect to the pulse labeling of proteins. Colchicine (10 microM) does not block any step of the secretory protein transport, but when introduced before the pulse it decreases the transfer of labeled proteins from the rough endoplasmic reticulum to the Golgi area, suppressing their temporary accumulation in the Golgi area before any alteration of this organelle is detectable. Moreover, colchicine inhibits protein release only from the secretory granules formed in its presence because the peroxidase discharge is diminished 1 h after colchicine addition, and the secretion of newly synthesized proteins is strongly inhibited only when colchicine is introduced before secretory granule formation. Morphometric studies show that there is a great increase of secondary lysosomes, related to crinophagy, as early as 40-50 min after colchicine is added. However, changes in lysosomal enzymatic activities remained biochemically undetectable. We conclude that: (a) the labile microtubular system does not seem indispensable for protein transport in the rough endoplasmic reticulum-Golgi area but may facilitate this step, perhaps by maintaining the spatial organization of this area; and (b) in the lacrimal gland, colchicine inhibits protein release not by acting on the steps of secretion following the secretory granule formation, but by acting chiefly on the steps preceding secretory granule formation, perhaps by making the secretory granules formed in its presence incapable of discharging their content.     

Vasoactive intestinal polypeptide (VIP)-containing neurons: distribution throughout the brain of the chick (Gallus domesticus) with focus upon the lateral septal organ

The distribution of VIP-like perikarya and fibers was determined throughout the chick brain. The most rostral immunoreactive perikarya were found to be cerebrospinal fluid-contacting neurons in the pars medialis of the lateral septal organ. Additional data were presented supporting the idea that the lateral septal organ is another circumventricular organ within the brain of birds (Kuenzel and van Tienhoven 1982). A large group of immunoreactive perikarya was found in the lateral hypothalamic area and appeared continuous with immunoreactive neurons in the anterior medial and ventromedial hypothalamic nuclei (n). A few perikarya were located in the paraventricular hypothalamic n. A number of immunoreactive neurons were found within and about the infundibular and inferior hypothalamic n., none however was immunoreactive cerebrospinal fluid-contacting neurons. Immunoreactive perikarya were found predominantly in laminae 10–11 of the stratum griseum et fibrosum superficiale. A few scattered perikarya were found ventromedial to the n. tegmenti pedunculo-pontinus pars compacta and locus ceruleus. Some of the immunoreactivity was unusual, being very homogeneous within the cell body with little evidence of the material in the axon or dendrites. Perikarya were found in the central gray, n. intercollicularis, and area ventralis of Tsai. The most caudal structure showing immunoreactive neurons was the n. reticularis paragigantocellularis lateralis. Brain areas containing the most abundant immunoreactive fibers, listed from the rostral-most location, were found in the ventromedial region of the lobus parolfactorius and the lateral septal n. Continuing caudally, there were immunoreactive fibers within the periventricular hypothalamic n.; some of the fibers were found to travel for some distance parallel to the third ventricle. Dense immunoreactive fibers were found in the tractus cortico-habenularis et cortico-septalis, medial habenular n. and posterior and dorsal n. of the archistriatum. A number of areas had what appeared to be baskets of immunoreactive fibers (perhaps immunoreactive terminals) surrounding non-reactive perikarya. Brain areas containing terminals included the piriform cortex, area ventralis of Tsai, interpeduncular n., and specific regions of the stratum griseum et fibrosum superficiale. A very dense immunoreactivity occurred within the external zone of the median eminence, the dorsolateral parabrachial n., and n. tractus solitarii. Vasoactive intestinal polypeptide appears to be a useful peptide for defining the neuroanatomical constituents of the visceral forebrain in birds.     

Cholecystokinin-octapeptide like immunoreactivity in the area postrema of the rat and cat

The distribution of cholecystokinin-8 (CCK-8)-like immunoreactivity in the area postrema of the rat and cat was visualized using the peroxidase, antiperoxidase technique. In the rat the greatest amount of immunostaining occurred in peripheral regions of the area postrema at intermediate and rostral levels. Caudally, scattered immunoreactivity predominated. After colchicine treatment, numerous immunoreactive somata were observed throughout the area postrema. The cat area postrema had a different and more complex pattern of immunostaining than the rat. Moderate to dense accumulations of immunostaining occurred in the ventromedial region of the area postrema bordering the solitary tract and dorsal vagal nuclei. The central region of the area postrema possessed scattered amounts of immunoreactivity at rostral levels. Following colchicine treatment, no visible CCK-8-like immunoreactive cell bodies were observed in the cat area postrema. Results of the present investigation provide morphological evidence for the role of CCK-8 in cardiovascular regulation and satiety. The difference in the distribution of CCK-8 in the rat and cat suggest a possible role in the emetic reflex.     

免疫组织化学方法检测脑红蛋白在大鼠中枢神经系统的分布

目的 探讨脑红蛋白（NGB）基因在中枢神经系统中的分布。方法 用免疫组织化学ABC法研究了NGB蛋白在成年大鼠脑内的分布和定位。结果 NGB蛋白在成年大鼠脑中有非常广泛的表达。其分布区域包括大脑皮质,海马,丘脑和下丘脑的部分核团,脑桥及小脑,NGB免疫反应阳性物质定位于神经元的细胞质。结论 NGB蛋白在大鼠脑中有非常广泛的表达,提示NGB基因在中枢神经系统的功能活动中可能起重要作用。     

Characterization of Enkephalin Release from Rat Striatum

Abstract: Using antisera specific for methionine- and leucine-enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2–3% of the total tissue stores of enkephalin could be released by potassium depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium-stimulated release; tyr-arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short-term incubation periods used in these experiments.     

Thyrotropin-releasing hormone (TRH)-immunoreactive structures in the brain of the domestic mallard

Summary The distribution of immunoreactive thyrotropin-releasing hormone (TRH) in the central nervous system of the domestic mallard was studied by means of the peroxidase-antiperoxidase technique. After colchicine pretreatment, the highest number of TRH-immunoreactive perikarya was found in the parvocellular subdivision of the paraventricular nucleus and in the preoptic region; a smaller number of immunostained perikarya was observed in the lateral hypothalamic area and in the posterior medial hypothalamic nucleus. TRH-immunoreactive nerve fibers were detected throughout the hypothalamus, forming a dense network in the periventricular area, paraventricular nucleus, preoptic-suprachiasmatic region, and baso-lateral hypothalamic area. TRH-containing nerve fibers and terminals occurred in the organon vasculosum of the lamina terminalis and in the external zone of the median eminence in juxtaposition with hypophyseal portal vessels. Scattered fibers were also seen in the internal zone of the median eminence and in the rostral portion of the neural lobe. Numerous TRH-immunoreactive fibers were detected in extra-hypothalamic brain regions: the highest number of immunoreactive nerve fibers was found in the lateral septum, nucleus accumbens, olfactory tubercle, and parolfactory lobe. Moderate numbers of fibers were located in the basal forebrain, dorsomedial thalamic nuclei, hippocampus, interpeduncular nucleus, and the central gray of the mesencephalon. The present findings suggest that TRH may be involved in hypophysiotropic regulatory mechanisms and, in addition, may also act as neuromodulator or neurotransmitter in other regions of the avian brain.     

Synaptic relations of neurotensinergic neurons in the dorsal raphe nucleus

The ultrastructure and synaptic relations of neurotensinergic neurons in the rat dorsal raphe nucleus (DRN) were examined. The neurotensin-like immunoreactive (NT-LI) neurons in the DRN were fusiform or spherical. The NT-LI perikarya could only be detected in colchicine-treated animals whereas the immunoreactive axon terminals could only be found in the anirnals not treated with colchicine. Although many NT-LI dendrites received synapses from nonimmunoreactive axon terminals, the NT-LI perikarya received few synapses. NT-LI axon terminals also made synapses on nonimmunoreactive dendrites. Occasionally, synapses were found between the NT-LI axon terminals and NT-LI dendrites in the cases in which the animals were not treated with colchicine.