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Structural plasticity of nonneuronal cells in the hypothalamo-neurohypophyseal system: in the right place at the right time
Structural plasticity of nonneuronal cells in the hypothalamo-neurohypophyseal system: in the right place at the right time A.K. Salm,* A.E. Ayoub and B.E. Lally Department of Neurobiology and Anatomy, West Virginia University School of Medicine, P.O. Box 9128, Morgantown, WV 26506-9128, USA p Correspondence address: Tel.: þ 1-304-293-2435; fax: þ 1-304-293-8159. E-mail: [email protected](A.K.S.)
Contents 1. 2.
3.
Introduction: structural plasticity in the central nervous system (CNS) Structural plasticity of astrocytes in the HNS 2.1. HNS 2.2. Glial retraction 2.3. Correlates of glial retraction in the SON and posterior pituitary 2.4. Reorientation of astrocytes in the SON-VGL 2.5. Immunocytochemical studies 2.6. Role of GFAP messenger RNA in astrocyte shape changes 2.7. Proliferation of astrocytes in the activated SON and posterior pituitary 2.8. Breakdown of basal lamina and tenascin 2.9. Activity dependent plasticity of microglia in the activated HNS? Concluding remarks
Structural plasticity of astrocytes in the normally functioning brain is widespread. Two of the best-studied regions where structural plasticity occurs are the supraoptic nucleus (SON) and posterior pituitary. In this chapter, we review recent developments in our understanding of how this structural remodeling comes about, including new findings that show microglia also participate in structural plasticity of the SON. Other brain areas where structural plasticity of astrocytes has been demonstrated are also briefly introduced. 1. Introduction: structural plasticity in the central nervous system (CNS) As this volume attests, glial cells maintain the intracellular milieu of the CNS in diverse ways. Central to their ability to perform their regulatory functions is the capacity to be in Advances in Molecular and Cell Biology, Vol. 31, pages 181–198 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1
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‘the right place at the right time’. Whether removing dopamine from synaptic clefts, buffering extracellular potassium or providing glutamine to neighboring neurons, glial cells must first have their processes placed in strategic locations. Structural plasticity of glia, predominantly that of astrocytes, occurs throughout the nervous system (see Salm et al., 1998 for review). Sites where structural plasticity of astrocytes has been documented in normal brains include the hippocampus with long term potentiation induction (Wenzel et al., 1991), in the suprachiasmatic nucleus across the diurnal cycle (Lavialle and Serviere, 1993), in the cerebellar cortex with motor learning (Black et al., 1990; Anderson et al., 1994), in the arcuate nucleus of the rat (Garcia-Segura et al., 1994; Olmos et al., 1989) and monkey (Witkin et al., 1991), across changing estrogen levels, and extensively, in the hypothalamo-neurohypophyseal system (HNS) in response to diverse conditions leading to the synthesis and release of the peptides oxytocin (OX) and vasopressin (VP). A feature common to all of these examples is that the neurons in the vicinity are vigorously challenged to perform their specific functions, e.g., transmission of photic information (in the suprachiasmatic nucleus) or maintenance of fluid homeostasis (in the HNS). Given this, one might predict that similar changes would occur in any brain region where neurons can be selectively challenged. Structural plasticity of astrocytes is not stereotypic and varies with region. This is especially true with respect to glial coverage versus synaptic contacts. For example, in the cerebellar cortex, motor learning is accompanied by formation of synapses, an increase in astrocytic surface volume, and increased coverage of synaptic elements (Anderson et al., 1994; Jones and Greenough, 1996). Conversely, in the arcuate nucleus, a decrease in astrocytic coverage of neuronal somata is inversely related to synaptic coverage (Olmos et al., 1989). Many of the functional systems where structural plasticity of astrocytes has been found are in the hypothalamus (Salm et al., 1998). In fact, the hypothalamus might be prone to such changes due to the perpetual challenge of maintaining homeostasis. An enhanced degree of ‘neural nimbleness’ may be required to respond to sometimes rapid changes in the internal milieu. The hypothalamus is also experimentally convenient, with specific functions being performed by discrete, easily identifiable, nuclei. These features, in particular, have made the HNS especially attractive for the study of structural plasticity of glia. 2. Structural plasticity of astrocytes in the HNS 2.1. HNS The HNS consists primarily of magnocellular neuroendocrine cells (MNCs) in the hypothalamic paraventricular nucleus (PVN) and the supraoptic nucleus (SON). These cells send their long axons through the hypothalamus to terminate in the posterior pituitary. It also includes the nucleus circularis (Hatton, 1976; Tweedle and Hatton, 1977) as well as accessory MNCs nearby in the hypothalamus. Many studies have been done examining structural remodeling of astrocytes and neurons in the PVN, SON and posterior pituitary. Especially interested readers are encouraged to refer to a number of exhaustive reviews (Theodosis and Poulain, 1993; Hatton, 1997; Theodosis et al., 1998; Hatton, 1999). Structural plasticity of glia has been characterized most thoroughly in
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the SON and posterior pituitary. The SON is relatively homogeneous compared to the multiple subnuclei described for the PVN (Armstrong et al., 1980). MNCs produce OX and VP, as well as other peptides colocalized with OX and VP (Levin and Sawchenko, 1993). The function of OX is to produce the smooth muscle contractions of parturition, lactation and orgasm, whereas VP acts at the kidneys to promote water reabsorption at the renal tubules in response to increased plasma or extracellular osmolality or hemorrhage. Therefore ‘activated’ MNCs can be studied in the HNS of animals who are recently postparturient or lactating, or animals who have been dehydrated via osmotic challenges. Dehydration regimens used have included prolonged water deprivation, chronic substitution of drinking water with 1.5– 2.0% saline solutions, or acutely, injection of hypertonic sodium chloride (Beagley and Hatton, 1992). However, other activating stimuli include factors surrounding expression of maternal behaviors (Salm et al., 1988) and restraint stress (Miyata et al., 1994). Since normally cycling female rats experience fluctuations in estrogen and other hormones that may affect astrocytes (Stone et al., 1997—see also chapter by Melcangi et al.), all of our work described below has used male rats. Dehydration is induced by 2% saline administration for varying time periods. Rehydration uses the same manipulation, followed by access to normal drinking water for varying times. The SON is populated by two distinct populations of astrocytes (Figs 1 and 2). One of these consists of the stellate cells that occupy the magnocellular region of the SON (the SON proper), where they envelope nearby MNCs. These astrocytes are immunopositive for the astrocytic marker glial fibrillary acidic protein. A second population consists of those astrocytes with cell bodies arrayed along the glial limitans ventral to the SON (SONVGL). Under basal conditions, the processes of SON-VGL astrocytes form a dense local meshwork. These cells also send long processes dorsally, sometimes as far as 500 mm, through the SON proper where they envelope the MNCs (Figs 1 and 2). These cells are also GFAP þ , but they are additionally reactive with antibodies to vimentin
Fig. 1. Light micrograph showing the SON adjacent to the optic chiasm (OC). The section has been stained with antibodies to glial fibrillary acidic protein (GFAP) and astrocyte fibers can be seen throughout the nucleus. The densely stained ventral glial limitans subjacent to the SON (SON-VGL) is seen most ventrally.
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Fig. 2. High-power micrograph of the SON from a control animal that has been stained for GFAP. A dense meshwork of fibers is seen throughout the nucleus, many of which are emanating dorsally from the SON-VGL. Asterisks denote regions where MNCs can be seen in relief.
(Bonfanti et al., 1993) and taurine (Decavel and Hatton, 1995). In the SON, MNC somata and dendrites are segregated, with MNCs most dorsal in the nucleus (arrow in Fig. 3). SON dendrites course as a group ventrolateral to the somata (DZ in Fig. 3), and therefore can be easily studied (Armstrong et al., 1982).
2.2. Glial retraction Magnocellular neurons of the SON are tightly packed together, and under basal conditions this space is occupied by astrocyte processes. Modney and Hatton (1989) estimated that 84% of MNC membrane in the SON is covered by glial processes under basal conditions. In 1976, Charles Tweedle, Glenn Hatton and colleagues at Michigan State University began publishing a series of electron microscopic studies describing remarkable changes in the dehydration-activated HNS (Tweedle and Hatton, 1976). Shortly thereafter, Dionysia Theodosis, Dominique Poulain and colleagues at INSERM in Bordeaux reported similar observations in lactating rats (Theodosis et al., 1981; Theodosis and Poulain, 1984). Both groups quantified a reduction in glial coverage of neuronal elements with activation that led to increased direct apposition of the membranes of somata and dendrites in the SON (although ‘direct appositions’ were first noticed in a report from Lafarga et al., 1975). Tweedle and Hatton further reported a reduction in glial envelopment of axon terminals in the posterior pituitary which accompanied activation of the HNS (Tweedle and Hatton, 1980a,b). They hypothesized that the changes in the activated SON and posterior pituitary were due to ‘glial retraction’ whereby the astrocytes actively retracted their processes from around the neurons and axon terminals, and then
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Fig. 3. Light level micrographs of 1 mm thick, plastic embedded, toluidine blue stained sections depicting the organization of the SON and a dehydration associated decrease in SON-VGL thickness. (A) Tissue from a normally hydrated male rat. Magnocellular neurons (open arrow) can be seen at the top of the picture, overlying the dendritic zone (DZ), where many dendrites can be seen in cross-section. Astrocyte cell bodies comprising the VGL are arrayed along the dotted line (p). SAS: subarachnoid space. (B) Tissue from a 9-day dehydrated rat. Note the reduction in thickness of the VGL, delineated by carets. This enables MNCs to be in close proximity to the SAS. Magnification bars ¼ 10 mm (after Bobak and Salm, 1996).
reinserted them when stimulation ceased (Tweedle and Hatton, 1976). We now know that glial retraction occurs rapidly and can be detected by electron microscopy within as little as five hours following an injection of hypertonic saline (Beagley and Hatton, 1994). Remarkably, these changes are reversible following cessation of activation, i.e., adequate periods of rehydration or post weaning. In the region of the cell bodies, this enables the neurons to form direct appositions. Likewise, in the DZ of the SON, ventral to the somata, dendrites also become directly apposed and form bundles of up to 13 dendrites (Perlmutter et al., 1984, 1985; Salm et al., 1988). In the SON, the functional consequence of the reduction in astrocyte coverage is to synchronize neuronal activity. Physiological studies have shown that activation of OX and VP neurons leads to the emergence of distinct firing patterns for each type of neuron. In lactating rats, OX neurons change from a slow, continuous firing rate to synchronized, high frequency discharges that precede the milk ejection reflex. This in turn leads to a bolus of OX release and subsequent contraction of the mammary myoepithelial cells (Wakerley and Lincoln, 1973; Lincoln and Wakerley, 1975). Vasopressin neurons, when activated, change from a slow continuous firing pattern to synchronized phasic-bursting (Poulain and Wakerley, 1982). Glial retraction may promote these changes in several ways. One is through the formation of novel synaptic contacts or ‘double synapses’ where one presynaptic profile forms synapses with two or more post-synaptic dendrites, somata, or both (Tweedle and Hatton, 1984). Under basal conditions, the majority of synapses onto
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MNC somata are single synapses. However, with 10 days of 2% saline administration there is a significant increase in the amount of somatic membrane contacted by double synapses. A concomitant, approximately equal, decrease in amount of membrane contacted by single synapses suggests that the retraction of a glial process allows double synapses to be formed from single synapses (Modney and Hatton, 1989). In the DZ, the appearance of bundles is accompanied by formation of gap junctions, and this too would serve to synchronize the activity of coupled neurons (Andrew et al., 1981). In addition to these changes that directly coordinate the MNCs, the absence of astrocyte processes where they normally exist leads to changes in the extracellular environment that heighten MNC excitability. The lack of astrocytes to buffer potassium (Tang et al., 1980) and glutamate (Oliet et al., 2001) is also excitatory to MNCs. In both the SON (Deleuze et al., 1998) and posterior pituitary (Miyata et al., 1997), the retraction of those astrocyte processes that contain taurine, an inhibitory amino acid, would also serve to enhance MNC excitability. In the posterior pituitary, axons and axon terminals are normally engulfed by the cytoplasm of pituicytes, which separates the terminals from fenestrations in the capillary bed of the pituitary. With dehydration, far more terminals are directly apposed to these portals to the general circulation (Tweedle and Hatton, 1980a,b). This too is reversible with rehydration or postweaning. Hence, retraction of pituicytes in the neural lobe mirrors astrocyte plasticity in the SON and allows OX and VP access to the circulation.
2.3. Correlates of glial retraction in the SON and posterior pituitary Our laboratory and others have been engaged in exploring factors surrounding structural remodeling in the SON (see Salm, 2000 for review). Factors that we have investigated so far include reorientation of astrocytes, reentry of glial cells into the cell cycle, downregulation of morphoregulatory molecules, dissolution of the basal lamina, changes in the GFAP, concomitant changes in expression of GFAP message and changes in the population of resident microglia.
2.4. Reorientation of astrocytes in the SON-VGL We have carried out extensive electron microsopic investigation of changes in SONVGL astrocytes in the activated SON (Bobak and Salm, 1996; Salm and Bobak, 1999). From montages of the SON-VGL constructed at 1100 £ magnification we learned that SON activation is accompanied by a remarkable reorientation of these cells (Fig. 4). Under basal conditions they are oriented predominantly vertically, with processes extending up through the MNCs. With two days of 2% saline administration we see this population assume a 458 orientation relative to the pial surface. By 7 days of activation, the SON-VGL astrocytes are seen in a predominantly horizontal position. With 14 days of rehydration, astrocytes of the SON-VGL once again are seen extending vertically into the MNCs. Functionally, we believe that this reorientation is one means by which glial processes retract from around MNCs.
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Fig. 4. Left: electron micrograph montage of two (A and B) vertically oriented astrocytes in the SON-VGL of a normally hydrated rat. Right: Electron micrograph montage of a horizontally oriented astrocyte in the SON-VGL of a 9-day dehydrated rat. Astrocytes resume their vertical orientations upon rehydration. Magnification bars equal 5 mm (after Bobak and Salm, 1996).
A second significant finding of our electron microscopic studies was that the SON-VGL undergoes significant thinning when the SON is activated (Bobak and Salm, 1996; Fig. 3). The result of this is to permit the MNCs to abut very closely to the underlying subarachnoid space and cerebrospinal fluid (CSF). It is already known that molecules may pass from the CSF deeply into the SON-VGL (Brightman, 1965). We speculate that this thinning might facilitate even more communication between MNCs and the CSF. However, rigorous investigation of this remains to be done. Another consequence of reorientation of SONVGL astrocyte processes may be to permit a continued glial interface between an enlarged SON proper and the underlying CSF. It has been known for many years that MNCs hypertrophy when activated (Hatton and Walters, 1973; Modney and Hatton, 1989) and the overall result is a significantly enlarged nucleus. Recent measurements in our laboratory of the mediolateral extent of the SON have found it significantly wider with activation (Hawrylak and Salm, 1999). With rehydration, again, the SON-VGL returns to normal width and thickness (Bobak and Salm, 1996) and the SON regains its normal mediolateral dimensions (Hawrylak and Salm, 1999).
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2.5. Immunocytochemical studies We have applied immunocytochemical methods and antibodies to the astrocytespecific GFAP (Uyeda et al., 1972; Dahl and Bignami, 1973) to the SON and posterior pituitary (Salm et al., 1982, 1985). Because GFAP is a major constituent of the astrocyte cytoskeleton, and is specific to astrocytes, it seemed reasonable that changes in astrocyte morphology in the activated SON would be reflected by changes in this protein. Studies performed on SON tissue from lactating rats revealed a significant reduction in immunoreactivity for GFAP relative to female estrous controls (Salm et al., 1985), consistent with the idea that astrocytes retract their processes in the activated SON. In the posterior pituitary, we and others (Suess and Pliska, 1981; Salm et al., 1982) also demonstrated for the first time that pituicytes of the posterior pituitary were, as long suspected, specialized astrocytes, as they also contain GFAP. This observation was somewhat surprising since, under normal conditions, few intermediate filaments are seen in rat pituicytes when examined by electron microscopy (Tweedle and Hatton, 1980a,b). As seen with immunostaining for GFAP, pituicytes possess fewer, and more stubby, processes than do normal stellate astrocytes throughout the CNS. This hampered efforts to document changes in cell shape. However, Miyata and colleagues have recently found that these cells stain robustly with antibodies to microtubule-associated protein 2 (MAP2), and they have been able to verify process retraction in response to dehydration (Miyata et al., 1999; Matsunaga et al., 1999). Interestingly, astrocytes in the SON do not stain for this protein. Thus, the presence of MAP2 in pituicytes may play a unique role in enabling shape changes in that region. In recent years, we have revisited the impact of MNC activation on GFAP immunoreactivity in the SON using dehydration as the stimulus (Hawrylak et al., 1998). As with lactation, a significant reduction in GFAP immunoreactivity was found with seven days of 2% saline substitution for drinking water. We also assessed recovery of staining and observed robust immunoreactivity with seven days of reintroduction to drinking water. As was the case with the earlier lactation study (Salm et al., 1985), our impression was that there were fewer, and thinner astrocyte processes remaining in the activated state. We then applied stereologic methods to the tissue from the dehydration study and confirmed a reversible reduction in astrocyte surface volume, i.e., a reduction of mostly astrocyte processes, in the dehydrated state (Hawrylak et al., 1999; Fig. 5). 2.6. Role of GFAP messenger RNA in astrocyte shape changes In two separate studies, we have found that reductions in GFAP immunoreactivity accompany activation of the SON (Fig. 5). One question that persists is whether there are commensurate changes in the GFAP mRNA that also occur, reflecting regulation of GFAP at the molecular level by events surrounding MNC activation. To answer this question, we have constructed a probe to GFAP mRNA from a plasmid containing linearized GFAP cDNA probe (Chen and Liem, 1994) to perform in situ hybridization on tissue from control, 2 and 7 day 2% saline administered, and 21 day rehydrated rats (Lally et al., 2001; Fig. 6). It appears that the GFAP mRNA in SON astrocytes is as labile as the protein derived from it. In normally hydrated control animals, there is a relatively low-level of
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Fig. 5. Micrographs of GFAP immunostaining in the SON of: (A) Control, (B) 6-day dehydrated, and (C) 6-day dehydrated/6-day rehydrated male rats. There is a significant reduction of immunoreactivity in the activated SON (B), which is reversible with the cessation of stimulation (C) (after Hawrylak et al., 1999).
message being expressed. The level of message then increases gradually at 2 days of activation to a peak at 7 days of saline treatment. GFAP mRNA expression in the SON of rats given saline for 7 days and then allowed to rehydrate for 21 days is essentially back to control levels in the majority, but not all, of the animals in this group. Hence, there appears to be an inverse relationship between message expression and protein expression—at least as seen with immunocytochemistry. These data suggest a feedback mechanism exists that upregulates message in response to decreased protein levels. The increase in message seen at 7 days of dehydration may represent a ‘stockpiling’, whereby the cell becomes prepared to produce protein at a rapid rate upon cessation of SON activation. Indeed, we have seen in our rehydration studies that at 7 days of rehydration, there is an immense, albeit not fully organized, network of immunoreactive astrocyte processes in the SON. The precise mechanisms of the relationship between GFAP and its mRNA in this population of astrocytes remains to be elucidated.
2.7. Proliferation of astrocytes in the activated SON and posterior pituitary One of the earliest observations in the literature of glial plasticity was that of increased glial numbers in the cerebral cortices of rats housed in enriched environments (Altman and Das, 1964; Diamond et al., 1966). Murray (1968) and Paterson and LeBlond (1977) investigated the possibility that glial cells also proliferate in the SON of male rats given 1.5% saline solutions in lieu of drinking water. Both studies found that a significant proliferation of nonneuronal cells accompanied activation of the SON. Using triple fluorescence labeling, we revisited this issue in the posterior pituitary with antibodies to bromodeoxyuridine (BrdU), GFAP, and the DNA marker DAPI (Murugaiyan and Salm, 1995). In the posterior pituitary, we found a significant increase in proliferated pituicytes by 9 days of saline treatment, but a trend was already evident already after 3 days.
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Fig. 6. Low-magnification micrographs depicting in situ hybridization for GFAP mRNA in the SONs of hydrated, 2- and 7-day dehydrated, and 7-day dehydrated/21-day rehydrated rats. Compare with Fig. 5 and note that during dehydration, when GFAP immunostaining is low, GFAP mRNA expression is high. Arrows delineate the boundaries of the nucleus.
With rehydration, numbers of BrdU þ pituicytes declined to control levels in 7 days. An unexpected observation in this study was that cells captured in the process of dividing exhibited only thin perisomatic cytoplasm and usually only two processes. Pituicytes that were not labeled with BrdU exhibited the usual array of short stubby processes. From this
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we concluded that dividing pituicytes temporarily lose their processes and that this may underlie some glial process retraction in the neural lobe. Since it has been established that SON MNCs are capable of dendritic release of OX and VP (Pow and Morris, 1989; Neumann et al., 1993), we have also investigated whether either peptide might have mitogenic effects on astroglia in culture (Lucas and Salm, 1995). A significant effect of OX was established for both hypothalamic and cortical astroglia, whereas VP only had an effect on cortical astroglia. Thus, it seems that OX could be a signal for that portion of structural remodeling attributable to reentry into the cell cycle of SON astrocytes. In this regard, Theodosis et al. (1986) have also presented evidence that OX is an activating signal in the structural remodeling of the SON.
2.8. Breakdown of basal lamina and tenascin In the course of our electron microscopic studies, we observed an intricate and extensive basal lamina in the dense array of glial processes in the SON-VGL (Salm and Bobak, 1999). It is well established that basal lamina and its associated molecules play a role in maintenance of cell polarity (Ojakian and Schwimmer, 1994; Klein et al., 1998). We therefore carried out a stereological study of the basal lamina across activation states in the SON-VGL. A significant reduction in basal lamina occurred with only two days of 2% saline substitution. With longer treatment, the extent of the basal lamina was progressively reduced, until it was virtually absent in the D7 group. With 9 days of access to normal drinking water, the basal lamina returned. It is interesting that the significant reduction in basal lamina at D2 precedes the findings of significant reorientation (Bobak and Salm, 1996), although trends in this direction are evident at D2. This is consistent with the idea that basal lamina reduction is permissive for structural plasticity (see chapter by Mercier and Hatton). Another contributing signal for structural remodeling in the SON-VGL may be the breakdown of the morphoregulatory molecule tenascin. Tenascin, a 210 – 220 kDa glycoprotein, has been implicated in neurite outgrowth regulation and boundary formation during development (Erickson and Lightner, 1988; Steindler et al., 1988). This molecule has received attention both from our group (Singleton and Salm, 1996) and Theodosis et al. (1997) as a possible regulator of plasticity in the SON. Painstaking cell cultures of tissue dissected solely from the SON revealed that tenascin is manufactured and secreted by SON astrocytes. Double labeling of tissue sections with antibodies to tenascin and GFAP revealed that in the SON-VGL, tenascin is only detectable in the residing astrocytes, and that immunoreactivity was reduced in tissue sections from six-day dehydrated animals. Protein biochemistry and Western blots further revealed that the reduction in the highmolecular weight forms was accompanied by the appearance of low 50– 60 kDa proteins, which were also immunoreactive to tenascin antibodies. With 6 days of rehydration, these smaller proteins disappeared while the larger form was again expressed. Thus, it appears that factors surrounding dehydration lead to the breakdown of tenascin. We speculate that the disappearance of tenascin is also permissive for the reorientation of the SON-VGL astrocytes that occurs with dehydration.
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2.9. Activity dependent plasticity of microglia in the activated HNS? A second population of nonneuronal cells that has thus far received only minor attention in the SON is the microglia. Mander and Morris (1995) investigated the presence of microglia in the SON with a panel of antibodies including OX-42, F4/80, ED2, OX-6 and OX-18 to demonstrate that, indeed, there are ramified and perivascular microglia in the SON. However, these antibodies only labeled relatively few microglia in the SON. This was our experience as well, therefore we used the isolectin-B4 method (Streit, 1990) for visualizing microglia in the SON. By this method, we can visualize a substantial population of microglia in the SON. They are predominantly found directly apposed to, or very near, the capillaries in the SON proper. Their numbers are small in the SON-VGL where they only appear at the pial surface (Ayoub and Salm, 2003; Fig. 7). We have recently investigated the impact of the dehydration stimulus on these cells. With normal hydration, the SON is populated by predominantly ramified microglia, and many of these appear to be astride the numerous capillaries in the nucleus (Fig. 8). With 2 days of dehydration, an additional population of hypertrophied microglia are seen in the nucleus. By 7 days, there are again significant numbers of hypertrophied microglia, but also a significant number of yet a third population of ameboid cells (Fig. 8). With rehydration, the population of hypertrophied and ameboid microglia disappear, and overall numbers return to normal levels. The role of microglia in the SON under differing activation states is unknown. They may play a role in removing breakdown products of tenascin or other components of the basal lamina. Pow et al. (1989) have shown with OX-42 antibodies and electron microscopy that there is a substantial (19%) population of microglia in the posterior pituitary. Here microglia appear to phagocytose peptidergic axon terminals, and thus may play a role in the sculpting of neurosecretory terminals under normal conditions. The source of the hypertrophied and ameboid microglia population is currently unknown, as there is no commensurate decrease in numbers of ramified microglia, and overall numbers of microglia increase significantly by D7. Thus, it is not clear whether individual cells are undergoing structural plasticity, or whether the new population of hypertrophied and ameboid microglia are being attracted to the nucleus by factors surrounding remodeling. The breakdown of extracellular matrix may be one such factor. It is tempting to speculate that at least some of the proliferation of nonneuronal cells previously reported in the SON (Murray, 1968; Paterson and LeBlond, 1977) includes microglia. However, we have only seen the occasional microglial cell in the SON in the act of dividing. The appearance of hypertrophied microglia at D2, prior to the finding of ameboid cells in the SON at D7 suggests that at least some cells undergo morphological transformation in the activated
Fig. 7. Low-magnification micrographs of the SONs of Control and 2-day dehydrated rats stained with isolectin B-4 to visualize microglia. The SON of the control rat exhibits light staining of ramified cells. The D2 section exhibit darker staining reflective of increased numbers of hypertrophied microglia. Thin arrows point to ramified microglia. Thicker arrows point to hypertrophied cells. OC: optic chiasm. p denotes capillaries. Stars indicate regions where MNCs are located.
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Fig. 8. High-magnification micrographs depicting three microglial phenotypes in the SON. (A) Ramified microglia: the thin arrow points to a ramified cell associated with a capillary. (B) The thick arrows point to hypertrophied microglia, the thinner arrow points to a ramified cell. (C) The thick arrow points to an ameboid microglial cell. Note the numerous filopodia emanating from this cell (after Ayoub and Salm, 2003).
SON. Further work is needed to establish that this is indeed an example of activitydependent plasticity of microglia under nonpathological conditions. 3. Concluding remarks Structural plasticity of astrocytes has been documented in normal brains in the hippocampus with LTP induction, in the suprachiasmatic nucleus across the diurnal cycle, in the cerebellar cortex with motor learning, in the arcuate nucleus of the rat and monkey, across changing estrogen levels, and extensively, in the HNS in response to diverse conditions leading to the synthesis and release of peptide hormones. A feature common to all of these examples is that the neurons in the vicinity are vigorously challenged to perform their specific functions. Given this, one might predict that similar changes would occur in any brain region where neurons can be selectively challenged. It is therefore of key interest to explore factors surrounding structural remodeling. Factors that we have investigated so far in the SON of the hypothalamus include reorientation of astrocytes, reentry of glial cells into the cell cycle, downregulation of morphoregulatory molecules, dissolution of the basal lamina, changes in the GFAP, concomitant changes in expression of GFAP message and changes in the population of resident microglia. Acknowledgements This work was supported by NSF IBN 9109827 and 951457. The authors thank Patricia Dickerson for critically reading the manuscript.
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Contents 1. 2.
3.
Introduction: structural plasticity in the central nervous system (CNS) Structural plasticity of astrocytes in the HNS 2.1. HNS 2.2. Glial retraction 2.3. Correlates of glial retraction in the SON and posterior pituitary 2.4. Reorientation of astrocytes in the SON-VGL 2.5. Immunocytochemical studies 2.6. Role of GFAP messenger RNA in astrocyte shape changes 2.7. Proliferation of astrocytes in the activated SON and posterior pituitary 2.8. Breakdown of basal lamina and tenascin 2.9. Activity dependent plasticity of microglia in the activated HNS? Concluding remarks
Structural plasticity of astrocytes in the normally functioning brain is widespread. Two of the best-studied regions where structural plasticity occurs are the supraoptic nucleus (SON) and posterior pituitary. In this chapter, we review recent developments in our understanding of how this structural remodeling comes about, including new findings that show microglia also participate in structural plasticity of the SON. Other brain areas where structural plasticity of astrocytes has been demonstrated are also briefly introduced. 1. Introduction: structural plasticity in the central nervous system (CNS) As this volume attests, glial cells maintain the intracellular milieu of the CNS in diverse ways. Central to their ability to perform their regulatory functions is the capacity to be in Advances in Molecular and Cell Biology, Vol. 31, pages 181–198 q 2004 Elsevier B.V. All rights of reproduction in any form reserved. ISBN: 0-444-51451-1
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‘the right place at the right time’. Whether removing dopamine from synaptic clefts, buffering extracellular potassium or providing glutamine to neighboring neurons, glial cells must first have their processes placed in strategic locations. Structural plasticity of glia, predominantly that of astrocytes, occurs throughout the nervous system (see Salm et al., 1998 for review). Sites where structural plasticity of astrocytes has been documented in normal brains include the hippocampus with long term potentiation induction (Wenzel et al., 1991), in the suprachiasmatic nucleus across the diurnal cycle (Lavialle and Serviere, 1993), in the cerebellar cortex with motor learning (Black et al., 1990; Anderson et al., 1994), in the arcuate nucleus of the rat (Garcia-Segura et al., 1994; Olmos et al., 1989) and monkey (Witkin et al., 1991), across changing estrogen levels, and extensively, in the hypothalamo-neurohypophyseal system (HNS) in response to diverse conditions leading to the synthesis and release of the peptides oxytocin (OX) and vasopressin (VP). A feature common to all of these examples is that the neurons in the vicinity are vigorously challenged to perform their specific functions, e.g., transmission of photic information (in the suprachiasmatic nucleus) or maintenance of fluid homeostasis (in the HNS). Given this, one might predict that similar changes would occur in any brain region where neurons can be selectively challenged. Structural plasticity of astrocytes is not stereotypic and varies with region. This is especially true with respect to glial coverage versus synaptic contacts. For example, in the cerebellar cortex, motor learning is accompanied by formation of synapses, an increase in astrocytic surface volume, and increased coverage of synaptic elements (Anderson et al., 1994; Jones and Greenough, 1996). Conversely, in the arcuate nucleus, a decrease in astrocytic coverage of neuronal somata is inversely related to synaptic coverage (Olmos et al., 1989). Many of the functional systems where structural plasticity of astrocytes has been found are in the hypothalamus (Salm et al., 1998). In fact, the hypothalamus might be prone to such changes due to the perpetual challenge of maintaining homeostasis. An enhanced degree of ‘neural nimbleness’ may be required to respond to sometimes rapid changes in the internal milieu. The hypothalamus is also experimentally convenient, with specific functions being performed by discrete, easily identifiable, nuclei. These features, in particular, have made the HNS especially attractive for the study of structural plasticity of glia. 2. Structural plasticity of astrocytes in the HNS 2.1. HNS The HNS consists primarily of magnocellular neuroendocrine cells (MNCs) in the hypothalamic paraventricular nucleus (PVN) and the supraoptic nucleus (SON). These cells send their long axons through the hypothalamus to terminate in the posterior pituitary. It also includes the nucleus circularis (Hatton, 1976; Tweedle and Hatton, 1977) as well as accessory MNCs nearby in the hypothalamus. Many studies have been done examining structural remodeling of astrocytes and neurons in the PVN, SON and posterior pituitary. Especially interested readers are encouraged to refer to a number of exhaustive reviews (Theodosis and Poulain, 1993; Hatton, 1997; Theodosis et al., 1998; Hatton, 1999). Structural plasticity of glia has been characterized most thoroughly in
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the SON and posterior pituitary. The SON is relatively homogeneous compared to the multiple subnuclei described for the PVN (Armstrong et al., 1980). MNCs produce OX and VP, as well as other peptides colocalized with OX and VP (Levin and Sawchenko, 1993). The function of OX is to produce the smooth muscle contractions of parturition, lactation and orgasm, whereas VP acts at the kidneys to promote water reabsorption at the renal tubules in response to increased plasma or extracellular osmolality or hemorrhage. Therefore ‘activated’ MNCs can be studied in the HNS of animals who are recently postparturient or lactating, or animals who have been dehydrated via osmotic challenges. Dehydration regimens used have included prolonged water deprivation, chronic substitution of drinking water with 1.5– 2.0% saline solutions, or acutely, injection of hypertonic sodium chloride (Beagley and Hatton, 1992). However, other activating stimuli include factors surrounding expression of maternal behaviors (Salm et al., 1988) and restraint stress (Miyata et al., 1994). Since normally cycling female rats experience fluctuations in estrogen and other hormones that may affect astrocytes (Stone et al., 1997—see also chapter by Melcangi et al.), all of our work described below has used male rats. Dehydration is induced by 2% saline administration for varying time periods. Rehydration uses the same manipulation, followed by access to normal drinking water for varying times. The SON is populated by two distinct populations of astrocytes (Figs 1 and 2). One of these consists of the stellate cells that occupy the magnocellular region of the SON (the SON proper), where they envelope nearby MNCs. These astrocytes are immunopositive for the astrocytic marker glial fibrillary acidic protein. A second population consists of those astrocytes with cell bodies arrayed along the glial limitans ventral to the SON (SONVGL). Under basal conditions, the processes of SON-VGL astrocytes form a dense local meshwork. These cells also send long processes dorsally, sometimes as far as 500 mm, through the SON proper where they envelope the MNCs (Figs 1 and 2). These cells are also GFAP þ , but they are additionally reactive with antibodies to vimentin
Fig. 1. Light micrograph showing the SON adjacent to the optic chiasm (OC). The section has been stained with antibodies to glial fibrillary acidic protein (GFAP) and astrocyte fibers can be seen throughout the nucleus. The densely stained ventral glial limitans subjacent to the SON (SON-VGL) is seen most ventrally.
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Fig. 2. High-power micrograph of the SON from a control animal that has been stained for GFAP. A dense meshwork of fibers is seen throughout the nucleus, many of which are emanating dorsally from the SON-VGL. Asterisks denote regions where MNCs can be seen in relief.
(Bonfanti et al., 1993) and taurine (Decavel and Hatton, 1995). In the SON, MNC somata and dendrites are segregated, with MNCs most dorsal in the nucleus (arrow in Fig. 3). SON dendrites course as a group ventrolateral to the somata (DZ in Fig. 3), and therefore can be easily studied (Armstrong et al., 1982).
2.2. Glial retraction Magnocellular neurons of the SON are tightly packed together, and under basal conditions this space is occupied by astrocyte processes. Modney and Hatton (1989) estimated that 84% of MNC membrane in the SON is covered by glial processes under basal conditions. In 1976, Charles Tweedle, Glenn Hatton and colleagues at Michigan State University began publishing a series of electron microscopic studies describing remarkable changes in the dehydration-activated HNS (Tweedle and Hatton, 1976). Shortly thereafter, Dionysia Theodosis, Dominique Poulain and colleagues at INSERM in Bordeaux reported similar observations in lactating rats (Theodosis et al., 1981; Theodosis and Poulain, 1984). Both groups quantified a reduction in glial coverage of neuronal elements with activation that led to increased direct apposition of the membranes of somata and dendrites in the SON (although ‘direct appositions’ were first noticed in a report from Lafarga et al., 1975). Tweedle and Hatton further reported a reduction in glial envelopment of axon terminals in the posterior pituitary which accompanied activation of the HNS (Tweedle and Hatton, 1980a,b). They hypothesized that the changes in the activated SON and posterior pituitary were due to ‘glial retraction’ whereby the astrocytes actively retracted their processes from around the neurons and axon terminals, and then
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Fig. 3. Light level micrographs of 1 mm thick, plastic embedded, toluidine blue stained sections depicting the organization of the SON and a dehydration associated decrease in SON-VGL thickness. (A) Tissue from a normally hydrated male rat. Magnocellular neurons (open arrow) can be seen at the top of the picture, overlying the dendritic zone (DZ), where many dendrites can be seen in cross-section. Astrocyte cell bodies comprising the VGL are arrayed along the dotted line (p). SAS: subarachnoid space. (B) Tissue from a 9-day dehydrated rat. Note the reduction in thickness of the VGL, delineated by carets. This enables MNCs to be in close proximity to the SAS. Magnification bars ¼ 10 mm (after Bobak and Salm, 1996).
reinserted them when stimulation ceased (Tweedle and Hatton, 1976). We now know that glial retraction occurs rapidly and can be detected by electron microscopy within as little as five hours following an injection of hypertonic saline (Beagley and Hatton, 1994). Remarkably, these changes are reversible following cessation of activation, i.e., adequate periods of rehydration or post weaning. In the region of the cell bodies, this enables the neurons to form direct appositions. Likewise, in the DZ of the SON, ventral to the somata, dendrites also become directly apposed and form bundles of up to 13 dendrites (Perlmutter et al., 1984, 1985; Salm et al., 1988). In the SON, the functional consequence of the reduction in astrocyte coverage is to synchronize neuronal activity. Physiological studies have shown that activation of OX and VP neurons leads to the emergence of distinct firing patterns for each type of neuron. In lactating rats, OX neurons change from a slow, continuous firing rate to synchronized, high frequency discharges that precede the milk ejection reflex. This in turn leads to a bolus of OX release and subsequent contraction of the mammary myoepithelial cells (Wakerley and Lincoln, 1973; Lincoln and Wakerley, 1975). Vasopressin neurons, when activated, change from a slow continuous firing pattern to synchronized phasic-bursting (Poulain and Wakerley, 1982). Glial retraction may promote these changes in several ways. One is through the formation of novel synaptic contacts or ‘double synapses’ where one presynaptic profile forms synapses with two or more post-synaptic dendrites, somata, or both (Tweedle and Hatton, 1984). Under basal conditions, the majority of synapses onto
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MNC somata are single synapses. However, with 10 days of 2% saline administration there is a significant increase in the amount of somatic membrane contacted by double synapses. A concomitant, approximately equal, decrease in amount of membrane contacted by single synapses suggests that the retraction of a glial process allows double synapses to be formed from single synapses (Modney and Hatton, 1989). In the DZ, the appearance of bundles is accompanied by formation of gap junctions, and this too would serve to synchronize the activity of coupled neurons (Andrew et al., 1981). In addition to these changes that directly coordinate the MNCs, the absence of astrocyte processes where they normally exist leads to changes in the extracellular environment that heighten MNC excitability. The lack of astrocytes to buffer potassium (Tang et al., 1980) and glutamate (Oliet et al., 2001) is also excitatory to MNCs. In both the SON (Deleuze et al., 1998) and posterior pituitary (Miyata et al., 1997), the retraction of those astrocyte processes that contain taurine, an inhibitory amino acid, would also serve to enhance MNC excitability. In the posterior pituitary, axons and axon terminals are normally engulfed by the cytoplasm of pituicytes, which separates the terminals from fenestrations in the capillary bed of the pituitary. With dehydration, far more terminals are directly apposed to these portals to the general circulation (Tweedle and Hatton, 1980a,b). This too is reversible with rehydration or postweaning. Hence, retraction of pituicytes in the neural lobe mirrors astrocyte plasticity in the SON and allows OX and VP access to the circulation.
2.3. Correlates of glial retraction in the SON and posterior pituitary Our laboratory and others have been engaged in exploring factors surrounding structural remodeling in the SON (see Salm, 2000 for review). Factors that we have investigated so far include reorientation of astrocytes, reentry of glial cells into the cell cycle, downregulation of morphoregulatory molecules, dissolution of the basal lamina, changes in the GFAP, concomitant changes in expression of GFAP message and changes in the population of resident microglia.
2.4. Reorientation of astrocytes in the SON-VGL We have carried out extensive electron microsopic investigation of changes in SONVGL astrocytes in the activated SON (Bobak and Salm, 1996; Salm and Bobak, 1999). From montages of the SON-VGL constructed at 1100 £ magnification we learned that SON activation is accompanied by a remarkable reorientation of these cells (Fig. 4). Under basal conditions they are oriented predominantly vertically, with processes extending up through the MNCs. With two days of 2% saline administration we see this population assume a 458 orientation relative to the pial surface. By 7 days of activation, the SON-VGL astrocytes are seen in a predominantly horizontal position. With 14 days of rehydration, astrocytes of the SON-VGL once again are seen extending vertically into the MNCs. Functionally, we believe that this reorientation is one means by which glial processes retract from around MNCs.
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Fig. 4. Left: electron micrograph montage of two (A and B) vertically oriented astrocytes in the SON-VGL of a normally hydrated rat. Right: Electron micrograph montage of a horizontally oriented astrocyte in the SON-VGL of a 9-day dehydrated rat. Astrocytes resume their vertical orientations upon rehydration. Magnification bars equal 5 mm (after Bobak and Salm, 1996).
A second significant finding of our electron microscopic studies was that the SON-VGL undergoes significant thinning when the SON is activated (Bobak and Salm, 1996; Fig. 3). The result of this is to permit the MNCs to abut very closely to the underlying subarachnoid space and cerebrospinal fluid (CSF). It is already known that molecules may pass from the CSF deeply into the SON-VGL (Brightman, 1965). We speculate that this thinning might facilitate even more communication between MNCs and the CSF. However, rigorous investigation of this remains to be done. Another consequence of reorientation of SONVGL astrocyte processes may be to permit a continued glial interface between an enlarged SON proper and the underlying CSF. It has been known for many years that MNCs hypertrophy when activated (Hatton and Walters, 1973; Modney and Hatton, 1989) and the overall result is a significantly enlarged nucleus. Recent measurements in our laboratory of the mediolateral extent of the SON have found it significantly wider with activation (Hawrylak and Salm, 1999). With rehydration, again, the SON-VGL returns to normal width and thickness (Bobak and Salm, 1996) and the SON regains its normal mediolateral dimensions (Hawrylak and Salm, 1999).
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2.5. Immunocytochemical studies We have applied immunocytochemical methods and antibodies to the astrocytespecific GFAP (Uyeda et al., 1972; Dahl and Bignami, 1973) to the SON and posterior pituitary (Salm et al., 1982, 1985). Because GFAP is a major constituent of the astrocyte cytoskeleton, and is specific to astrocytes, it seemed reasonable that changes in astrocyte morphology in the activated SON would be reflected by changes in this protein. Studies performed on SON tissue from lactating rats revealed a significant reduction in immunoreactivity for GFAP relative to female estrous controls (Salm et al., 1985), consistent with the idea that astrocytes retract their processes in the activated SON. In the posterior pituitary, we and others (Suess and Pliska, 1981; Salm et al., 1982) also demonstrated for the first time that pituicytes of the posterior pituitary were, as long suspected, specialized astrocytes, as they also contain GFAP. This observation was somewhat surprising since, under normal conditions, few intermediate filaments are seen in rat pituicytes when examined by electron microscopy (Tweedle and Hatton, 1980a,b). As seen with immunostaining for GFAP, pituicytes possess fewer, and more stubby, processes than do normal stellate astrocytes throughout the CNS. This hampered efforts to document changes in cell shape. However, Miyata and colleagues have recently found that these cells stain robustly with antibodies to microtubule-associated protein 2 (MAP2), and they have been able to verify process retraction in response to dehydration (Miyata et al., 1999; Matsunaga et al., 1999). Interestingly, astrocytes in the SON do not stain for this protein. Thus, the presence of MAP2 in pituicytes may play a unique role in enabling shape changes in that region. In recent years, we have revisited the impact of MNC activation on GFAP immunoreactivity in the SON using dehydration as the stimulus (Hawrylak et al., 1998). As with lactation, a significant reduction in GFAP immunoreactivity was found with seven days of 2% saline substitution for drinking water. We also assessed recovery of staining and observed robust immunoreactivity with seven days of reintroduction to drinking water. As was the case with the earlier lactation study (Salm et al., 1985), our impression was that there were fewer, and thinner astrocyte processes remaining in the activated state. We then applied stereologic methods to the tissue from the dehydration study and confirmed a reversible reduction in astrocyte surface volume, i.e., a reduction of mostly astrocyte processes, in the dehydrated state (Hawrylak et al., 1999; Fig. 5). 2.6. Role of GFAP messenger RNA in astrocyte shape changes In two separate studies, we have found that reductions in GFAP immunoreactivity accompany activation of the SON (Fig. 5). One question that persists is whether there are commensurate changes in the GFAP mRNA that also occur, reflecting regulation of GFAP at the molecular level by events surrounding MNC activation. To answer this question, we have constructed a probe to GFAP mRNA from a plasmid containing linearized GFAP cDNA probe (Chen and Liem, 1994) to perform in situ hybridization on tissue from control, 2 and 7 day 2% saline administered, and 21 day rehydrated rats (Lally et al., 2001; Fig. 6). It appears that the GFAP mRNA in SON astrocytes is as labile as the protein derived from it. In normally hydrated control animals, there is a relatively low-level of
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Fig. 5. Micrographs of GFAP immunostaining in the SON of: (A) Control, (B) 6-day dehydrated, and (C) 6-day dehydrated/6-day rehydrated male rats. There is a significant reduction of immunoreactivity in the activated SON (B), which is reversible with the cessation of stimulation (C) (after Hawrylak et al., 1999).
message being expressed. The level of message then increases gradually at 2 days of activation to a peak at 7 days of saline treatment. GFAP mRNA expression in the SON of rats given saline for 7 days and then allowed to rehydrate for 21 days is essentially back to control levels in the majority, but not all, of the animals in this group. Hence, there appears to be an inverse relationship between message expression and protein expression—at least as seen with immunocytochemistry. These data suggest a feedback mechanism exists that upregulates message in response to decreased protein levels. The increase in message seen at 7 days of dehydration may represent a ‘stockpiling’, whereby the cell becomes prepared to produce protein at a rapid rate upon cessation of SON activation. Indeed, we have seen in our rehydration studies that at 7 days of rehydration, there is an immense, albeit not fully organized, network of immunoreactive astrocyte processes in the SON. The precise mechanisms of the relationship between GFAP and its mRNA in this population of astrocytes remains to be elucidated.
2.7. Proliferation of astrocytes in the activated SON and posterior pituitary One of the earliest observations in the literature of glial plasticity was that of increased glial numbers in the cerebral cortices of rats housed in enriched environments (Altman and Das, 1964; Diamond et al., 1966). Murray (1968) and Paterson and LeBlond (1977) investigated the possibility that glial cells also proliferate in the SON of male rats given 1.5% saline solutions in lieu of drinking water. Both studies found that a significant proliferation of nonneuronal cells accompanied activation of the SON. Using triple fluorescence labeling, we revisited this issue in the posterior pituitary with antibodies to bromodeoxyuridine (BrdU), GFAP, and the DNA marker DAPI (Murugaiyan and Salm, 1995). In the posterior pituitary, we found a significant increase in proliferated pituicytes by 9 days of saline treatment, but a trend was already evident already after 3 days.
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Fig. 6. Low-magnification micrographs depicting in situ hybridization for GFAP mRNA in the SONs of hydrated, 2- and 7-day dehydrated, and 7-day dehydrated/21-day rehydrated rats. Compare with Fig. 5 and note that during dehydration, when GFAP immunostaining is low, GFAP mRNA expression is high. Arrows delineate the boundaries of the nucleus.
With rehydration, numbers of BrdU þ pituicytes declined to control levels in 7 days. An unexpected observation in this study was that cells captured in the process of dividing exhibited only thin perisomatic cytoplasm and usually only two processes. Pituicytes that were not labeled with BrdU exhibited the usual array of short stubby processes. From this
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we concluded that dividing pituicytes temporarily lose their processes and that this may underlie some glial process retraction in the neural lobe. Since it has been established that SON MNCs are capable of dendritic release of OX and VP (Pow and Morris, 1989; Neumann et al., 1993), we have also investigated whether either peptide might have mitogenic effects on astroglia in culture (Lucas and Salm, 1995). A significant effect of OX was established for both hypothalamic and cortical astroglia, whereas VP only had an effect on cortical astroglia. Thus, it seems that OX could be a signal for that portion of structural remodeling attributable to reentry into the cell cycle of SON astrocytes. In this regard, Theodosis et al. (1986) have also presented evidence that OX is an activating signal in the structural remodeling of the SON.
2.8. Breakdown of basal lamina and tenascin In the course of our electron microscopic studies, we observed an intricate and extensive basal lamina in the dense array of glial processes in the SON-VGL (Salm and Bobak, 1999). It is well established that basal lamina and its associated molecules play a role in maintenance of cell polarity (Ojakian and Schwimmer, 1994; Klein et al., 1998). We therefore carried out a stereological study of the basal lamina across activation states in the SON-VGL. A significant reduction in basal lamina occurred with only two days of 2% saline substitution. With longer treatment, the extent of the basal lamina was progressively reduced, until it was virtually absent in the D7 group. With 9 days of access to normal drinking water, the basal lamina returned. It is interesting that the significant reduction in basal lamina at D2 precedes the findings of significant reorientation (Bobak and Salm, 1996), although trends in this direction are evident at D2. This is consistent with the idea that basal lamina reduction is permissive for structural plasticity (see chapter by Mercier and Hatton). Another contributing signal for structural remodeling in the SON-VGL may be the breakdown of the morphoregulatory molecule tenascin. Tenascin, a 210 – 220 kDa glycoprotein, has been implicated in neurite outgrowth regulation and boundary formation during development (Erickson and Lightner, 1988; Steindler et al., 1988). This molecule has received attention both from our group (Singleton and Salm, 1996) and Theodosis et al. (1997) as a possible regulator of plasticity in the SON. Painstaking cell cultures of tissue dissected solely from the SON revealed that tenascin is manufactured and secreted by SON astrocytes. Double labeling of tissue sections with antibodies to tenascin and GFAP revealed that in the SON-VGL, tenascin is only detectable in the residing astrocytes, and that immunoreactivity was reduced in tissue sections from six-day dehydrated animals. Protein biochemistry and Western blots further revealed that the reduction in the highmolecular weight forms was accompanied by the appearance of low 50– 60 kDa proteins, which were also immunoreactive to tenascin antibodies. With 6 days of rehydration, these smaller proteins disappeared while the larger form was again expressed. Thus, it appears that factors surrounding dehydration lead to the breakdown of tenascin. We speculate that the disappearance of tenascin is also permissive for the reorientation of the SON-VGL astrocytes that occurs with dehydration.
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2.9. Activity dependent plasticity of microglia in the activated HNS? A second population of nonneuronal cells that has thus far received only minor attention in the SON is the microglia. Mander and Morris (1995) investigated the presence of microglia in the SON with a panel of antibodies including OX-42, F4/80, ED2, OX-6 and OX-18 to demonstrate that, indeed, there are ramified and perivascular microglia in the SON. However, these antibodies only labeled relatively few microglia in the SON. This was our experience as well, therefore we used the isolectin-B4 method (Streit, 1990) for visualizing microglia in the SON. By this method, we can visualize a substantial population of microglia in the SON. They are predominantly found directly apposed to, or very near, the capillaries in the SON proper. Their numbers are small in the SON-VGL where they only appear at the pial surface (Ayoub and Salm, 2003; Fig. 7). We have recently investigated the impact of the dehydration stimulus on these cells. With normal hydration, the SON is populated by predominantly ramified microglia, and many of these appear to be astride the numerous capillaries in the nucleus (Fig. 8). With 2 days of dehydration, an additional population of hypertrophied microglia are seen in the nucleus. By 7 days, there are again significant numbers of hypertrophied microglia, but also a significant number of yet a third population of ameboid cells (Fig. 8). With rehydration, the population of hypertrophied and ameboid microglia disappear, and overall numbers return to normal levels. The role of microglia in the SON under differing activation states is unknown. They may play a role in removing breakdown products of tenascin or other components of the basal lamina. Pow et al. (1989) have shown with OX-42 antibodies and electron microscopy that there is a substantial (19%) population of microglia in the posterior pituitary. Here microglia appear to phagocytose peptidergic axon terminals, and thus may play a role in the sculpting of neurosecretory terminals under normal conditions. The source of the hypertrophied and ameboid microglia population is currently unknown, as there is no commensurate decrease in numbers of ramified microglia, and overall numbers of microglia increase significantly by D7. Thus, it is not clear whether individual cells are undergoing structural plasticity, or whether the new population of hypertrophied and ameboid microglia are being attracted to the nucleus by factors surrounding remodeling. The breakdown of extracellular matrix may be one such factor. It is tempting to speculate that at least some of the proliferation of nonneuronal cells previously reported in the SON (Murray, 1968; Paterson and LeBlond, 1977) includes microglia. However, we have only seen the occasional microglial cell in the SON in the act of dividing. The appearance of hypertrophied microglia at D2, prior to the finding of ameboid cells in the SON at D7 suggests that at least some cells undergo morphological transformation in the activated
Fig. 7. Low-magnification micrographs of the SONs of Control and 2-day dehydrated rats stained with isolectin B-4 to visualize microglia. The SON of the control rat exhibits light staining of ramified cells. The D2 section exhibit darker staining reflective of increased numbers of hypertrophied microglia. Thin arrows point to ramified microglia. Thicker arrows point to hypertrophied cells. OC: optic chiasm. p denotes capillaries. Stars indicate regions where MNCs are located.
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Fig. 8. High-magnification micrographs depicting three microglial phenotypes in the SON. (A) Ramified microglia: the thin arrow points to a ramified cell associated with a capillary. (B) The thick arrows point to hypertrophied microglia, the thinner arrow points to a ramified cell. (C) The thick arrow points to an ameboid microglial cell. Note the numerous filopodia emanating from this cell (after Ayoub and Salm, 2003).
SON. Further work is needed to establish that this is indeed an example of activitydependent plasticity of microglia under nonpathological conditions. 3. Concluding remarks Structural plasticity of astrocytes has been documented in normal brains in the hippocampus with LTP induction, in the suprachiasmatic nucleus across the diurnal cycle, in the cerebellar cortex with motor learning, in the arcuate nucleus of the rat and monkey, across changing estrogen levels, and extensively, in the HNS in response to diverse conditions leading to the synthesis and release of peptide hormones. A feature common to all of these examples is that the neurons in the vicinity are vigorously challenged to perform their specific functions. Given this, one might predict that similar changes would occur in any brain region where neurons can be selectively challenged. It is therefore of key interest to explore factors surrounding structural remodeling. Factors that we have investigated so far in the SON of the hypothalamus include reorientation of astrocytes, reentry of glial cells into the cell cycle, downregulation of morphoregulatory molecules, dissolution of the basal lamina, changes in the GFAP, concomitant changes in expression of GFAP message and changes in the population of resident microglia. Acknowledgements This work was supported by NSF IBN 9109827 and 951457. The authors thank Patricia Dickerson for critically reading the manuscript.
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