Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (Aß) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that Aß is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e., microglia), which culminates in neuronal damage and cognitive decline. To test this hypothesis, many in vitro systems have been established to study Aß-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale. METHODS: We used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic Aß or LPS and INFγ. RESULTS: We found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model that best resembles primary microglia. Surprisingly, synthetic Aß does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of Aß formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INFγ activate microglia and induce transcriptional changes that resemble many, but not all, aspects of the transcriptomic profiles of disease associated microglia (DAM) present in the AD brain. CONCLUSIONS: These results suggest that synthetic Aß treatment of innate immune cell cultures does not recapitulate transcriptional profiles observed in microglia from AD brains. In contrast, treating IMGL with LPS and INFγ induces transcriptional changes similar to those observed in microglia detected in AD brains.

Astrocytes and microglia differentially regulate trafficking of lymphocyte subsets across brain endothelial cells.

Feline brain endothelial cells (BECs), astrocytes, and microglia were combined in different configurations in a cell culture insert system to assess the effect of different cell types on the trafficking of peripheral blood mononuclear cell (PBMC) subsets in response to feline immunodeficiency virus (FIV). The addition of astrocytes to BECs significantly increased the adherence of PBMCs. This increase in adherence was suppressed by microglia, whereas microglia alone had no effect on PBMC adherence. FIV exposure of the glial cells did not alter PBMC adherence as compared to same configurations with untreated cells. All PBMC subsets showed some level of trafficking across the endothelial cell layer. The level of trafficking of monocytes and B cells was significantly increased if astrocytes were present. The presence of microglia with the astrocytes reduced transmigration across all PBMC subsets. FIV exposure of astrocytes significantly increased the percentage of CD8 T cell transmigration from 24% to 64% of the total CD4 and CD8 numbers. The presence of microglia significantly reversed the preferential trafficking of CD8 cells in the presence of astrocytes. The results suggested that interaction between the triad of endothelial cells, astrocytes, and microglia played an important, but varying, role in the trafficking of different PBMC subsets. In general, astrocytes had a positive effect on trafficking of PBMCs, while microglia had a suppressive effect. Effects of FIV on trafficking were largely restricted to increases seen in CD8 T cells and monocytes.

Development of neuronal sensitivity to toxins in cerebrospinal fluid from HIV-type 1-infected individuals.

HIV infection of the immature nervous system generally results in a rapid progression of neurological disease that cannot easily be explained by the severity of encephalitis, viral burden, systemic immune deficiency, or developmental changes in utero. Rather than the viral infection dictating disease progression, we explored the possibility that immature neurons might be particularly sensitive to toxins secreted in response to HIV. Primary cultures of rat cortical neurons were exposed to toxic cerebrospinal fluid (CSF) from HIV-infected individuals (CSF(tox)) and evaluated for changes in intracellular calcium and cell death. CSF(tox) had no detectable effect on early neurite outgrowth, calcium regulation, or cell death during the first few days in culture. Starting at Day 4, delayed increases in intracellular calcium appeared in response to CSF(tox). The magnitude of the delayed calcium rise and cell death increased with the age of the culture and correlated with the appearance of synaptophysin immunoreactive varicosities. A similar gradual development of sensitivity was seen during exposure of feline neurons to toxins generated by choroid plexus macrophages after exposure to feline immunodeficiency virus. The possibility that toxin sensitivity is dependent on the presence of synaptic activity is consistent with the rapid pathogenesis in the CNS seen during the first postnatal year. Emerging synaptic activity coupled with other factors such as high metabolic demand in the young nervous system may combine to increase the likelihood of calcium overload and neuronal dysfunction in response to HIV-associated toxins.

Infection of the choroid plexus by feline immunodeficiency virus.

The human, simian, and feline immunodeficiency viruses rapidly penetrate into the brain and trigger an inflammatory process that can lead to significant neurologic disease. However, the mechanisms that permit efficient trafficking of macrophage-tropic and the more neurotoxic lymphocytotropic isolates are still poorly understood. One potential source of virus entry may be the blood-CSF barrier provided by the choroid plexus. Infected cells are often detected within the choroid plexus but it is unclear whether this reflects trafficking cells or infection of the large macrophage population within the choroidal stroma. To address this issue, we cultured fetal feline choroid plexus and evaluated the ability of feline immunodeficiency virus (FIV) to establish a primary infection. Significant provirus was detected in macrophage-enriched choroid plexus cultures as well as in the choroid plexus of cats infected in vivo. FIV p24 antigen production in vitro was very low but detectable. Addition of a feline T-cell line to macrophages inoculated with FIV resulted in a dense clustering of the T cells over macrophages with dendritic cell-like morphologies and a robust productive infection. The direct infection of choroid plexus macrophages with FIV, the efficient transfer of the infection to T cells indicate that the choroid plexus can be a highly efficient site of viral infection and perhaps trafficking of both macrophage-tropic and T-cell-tropic viruses into the CNS.

Choroid plexus macrophages proliferate and release toxic factors in response to feline immunodeficiency virus.

Recent observations have suggested that lentiviruses stimulate the proliferation and activation of microglia. A similar effect within the dense macrophage population of the choroid plexus could have significant implications for trafficking of virus and inflammatory cells into the brain. To explore this possibility, we cultured fetal feline macrophages and examined their response to feline immunodeficiency virus (FIV) or the T-cell-derived protein, recombinant human CD40-ligand trimer (rhuCD40-L). The rhCD40-L was the most potent stimulus for macrophage proliferation, often inducing a dramatic increase in macrophage density. Exposure to FIV resulted in a small increase in the number of macrophages and macrophage nuclei labeled with bromodeoxyuridine. The increase in macrophage density after FIV infection also correlated with an increase in neurotoxic activity of the macrophage-conditioned medium. Starting at 16-18 weeks postinfection, well after the peak of viremia, a similar toxic activity was detected in cerebrospinal fluid (CSF) from FIV-infected cats. Toxicity in the CSF increased over time and was paralleled by strong CD18 staining of macrophages/microglia in the choroid plexus and adjacent parenchyma. These results suggest that lentiviral infection of the choroid plexus can induce a toxic inflammatory response that is fueled by local macrophage proliferation. Together with the observation of increasing toxic activity in the CSF and increased CD18 staining in vivo, these observations suggest that choroid plexus macrophages may contribute to an inflammatory cascade in the brain that progresses independently of systemic and CSF viral load.

Destabilization of neuronal calcium homeostasis by factors secreted from choroid plexus macrophage cultures in response to feline immunodeficiency virus.

The choroid plexus contains a major reservoir of macrophages poised for efficient delivery of virus and neurotoxins to the brain after infection by lentiviruses such as human or feline immunodeficiency virus (FIV). However, their contribution to neurotoxicity is poorly understood. Medium from FIV-infected, choroid plexus macrophages applied to cultured feline cortical neurons induced a small acute calcium rise followed by either a delayed calcium deregulation (41%) or swelling and bursting (23%). NMDA glutamate receptor blockade prevented the acute calcium increase and antagonists to the IP(3) receptor, voltage-gated calcium channels and sodium channels suppressed both the acute and late increases. Analysis of intracellular calcium recovery in toxin-treated neurons after a brief exposure to glutamate, revealed a decrease in the rate and extent of recovery. The apparent diverse pharmacological contributions to intracellular calcium destabilization may be due to the ability of macrophage toxins to interfere with recovery of intracellular calcium homeostasis.

A role for the cytoskeleton-associated protein palladin in neurite outgrowth.

The outgrowth of neurites is a critical step in neuronal maturation, and it is well established that the actin cytoskeleton is involved in this process. Investigators from our laboratory recently described a novel protein named palladin, which has been shown to play an essential role in organizing the actin cytoskeleton in cultured fibroblasts. We investigated the expression of palladin in the developing rat brain by Western blot and found that the E18 brain contained a unique variant of palladin that is significantly smaller (approximately 85 kDa) than the common form found in other developing tissues (90-92 kDa). Because the expression of a tissue-specific isoform suggests the possibility of a cell type-specific function, we investigated the localization and function of palladin in cultured cortical neurons. Palladin was found preferentially targeted to the developing axon but not the dendrites and was strongly localized to the axonal growth cone. When palladin expression was attenuated by transfection with antisense constructs in both the B35 neuroblastoma cell line and in primary cortical neurons, a reduction in the expression of palladin resulted in a failure of neurite outgrowth. These results implicate palladin as a critical component of the developing nervous system, with an important role in axonal extension.

Choline deficiency induces apoptosis in primary cultures of fetal neurons.

Treatment of rats with choline during brain development results in long-lasting enhancement of spatial memory whereas choline deficiency has the opposite effect. Changes in rates of apoptosis may be responsible. We previously demonstrated that choline deficiency induced apoptosis in PC12 cells and suggested that interruption of cell cycling due to a decrease in membrane phosphatidylcholine concentration was the critical mechanism. We now examine whether choline deprivation induces apoptosis in nondividing primary neuronal cultures of fetal rat cortex and hippocampus. Choline deficiency induced widespread apoptosis in primary neuronal cells, indicating that cells do not have to be dividing to be sensitive to choline deficiency. When switched to a choline-deficient medium, both types of cells became depleted of choline, phosphocholine and phosphatidylcholine, and in primary neurons neurite outgrowth was dramatically attenuated. Primary cells could be rescued from apoptosis by treatment with phosphocholine or lysophosphatidylcholine. As described previously for PC12 cells, an increase in ceramide (Cer) was associated with choline deficiency-induced apoptosis in primary neurons. The primary neuronal culture appears to be an excellent model to explore the mechanism whereby maternal dietary choline intake modulates apoptosis in the fetal brain.

Microglial proliferation in cortical neural cultures exposed to feline immunodeficiency virus.

Microglia are thought to play an important role in neurodegenerative changes due to infection with human or animal immunodeficiency viruses. Using feline immunodeficiency virus and cat neural cultures, we observed a dramatic increase in the accumulation of microglia from a basal rate of 5-7% day(-1) to 25-126% day(-1). Both live virus and heat-inactivated virus induced proliferation. Negligible proliferation was seen in purified microglial cultures. Conditioned medium from astrocytes or mixed neural cultures treated with feline immunodeficiency virus stimulated the proliferation of purified microglia. Disease progression may be facilitated by early non-infectious interactions of lentiviruses with neural tissue that promote the activation and proliferation of microglia.

Neurotoxicity of CSF from HIV-infected humans.

Approximately 15-20% of individuals infected with the human immunodeficiency virus will develop severe neurological disease. This may be due in part to virus-induced release of a number of putative neurotoxins. However, there is little information to predict which individuals will progress to dementia or the precise mechanisms that drive pathogenesis. In an effort to identify early markers of neurological disease progression we used an in vitro bioassay with rat cortical neurons to test for the presence of toxins in CSF from 40 HIV-infected humans with mild, minimal or no neurological disease. A subset of HIV-infected individuals was found to have significant toxic activity in CSF indicating that toxic factors may be circulating prior to the development of dementia. The toxicity was concentration dependent and due to a factor with a molecular mass of less than 30 kDa. Only a small proportion of the cell death appeared to be due to apoptosis. Neuronal toxicity was associated with a gradual accumulation of intracellular calcium in a subset of cortical neurons over a period of 1-2 h and in the absence of a significant acute response. Individuals with both high viral burden and high CSF toxicity were significantly more likely to have neurological symptoms. These initial analyses indicate that toxic factors are present in the CSF of HIV-infected patients that could serve as useful markers of neurological disease progression and provide insights into pathogenic mechanisms in vivo.

Functional activation of punch-cultured magnocellular neuroendocrine cells by glutamate receptor subtypes.

To provide a simple means to isolate and study the cellular functions of small groups of neurons, we developed a modified 'punch' culture procedure that facilitates acute and long-term in vitro physiological studies. Primary 'punch' cultures of magnocellular neuroendocrine cells (MNCs) from the supraoptic nucleus (SON) were established and the basic physiological effects of subtype-specific glutamate receptor agonists were characterized. MNCs from the punch cultures established a mature morphology in culture with extensive outgrowth of axons and varicosities. After 8 days, a single cultured SON punch produced an average of 10.0 +/- 2.1 pg AVP and contained an average of 222 +/- 53 vasopressin-neurophysin immunoreactive cells. Patch clamp recordings from MNCs demonstrated the presence of N-methyl-D-aspartate (NMDA)-sensitive and DL, alpha-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA)-receptors. Stimulation of metabotropic receptors with 1S,3R ACPD induced acute or gradual increases in intracellular calcium. NMDA, AMPA and metabotropic receptors all contributed to the secretion of vasopressin from the punch cultures with an agonist rank order potency of: NMDA (10 microM) > AMPA (1 microM) = 1S,3R ACPD (100 microM) > kainate (10 microM). This culture preparation should be useful for cellular studies of small groups of neuroendocrine and other cells.

Neurotoxicity of FIV and FIV envelope protein in feline cortical cultures.

The neurotoxic effects of the feline immunodeficiency virus (FIV) and FIV envelope proteins were measured in primary cultures of feline cortical neurons. Envelope protein from the FIV-PPR strain promoted neuronal swelling and death, whereas envelope protein from the FIV-34TF10 isolate produced intermediate or negligible toxicity. No effect was observed in control cultures treated with envelope protein from the Epstein-Barr virus. A concentration-effect curve showed that FIV-PPR protein produced maximal toxicity at 200 pM protein and decreased toxicity at higher concentrations, which is consistent with previous reports of the HIV-1 surface glycoprotein, gp120. These effects required the presence of low concentrations of glutamate. Using the natural host cells as targets, the effects of envelope protein and infectious virions were directly compared. All of the toxic activity could be attributed to non-infectious interactions between the viral envelope and target cells. Addition of 1 microM tetrodotoxin failed to block the effects of FIV-PPR in the presence of 20 microM glutamate. Toxicity would appear to involve two steps in which the envelope protein first sensitizes neurons through non-synaptic interactions (TTX insensitive) thereby setting the stage for enhanced synaptic activation via glutamate receptors (TTX sensitive).

Kindled seizures increase metabotropic glutamate receptor expression and function in the rat supraoptic nucleus.

The spread of experimentally kindled seizures in rats results in sustained increases in plasma vasopressin (VP) and VP mRNA in the supraoptic nucleus (SON). These increases provide an excellent example of the pathological plasticity that can develop in normal cells exposed to recurrent seizure activity. To test whether this plasticity might be due in part to changes in metabotropic glutamate receptors (mGluRs), we examined mGluR mRNA expression in the SON 1 month after stage 5 amygdala kindling. Three mGluR subtypes were detected by in situ hybridization in the SON in the following relative levels: mGluR3 > mGluR1 > mGluR7. Both mGluR1 and mGluR3 mRNAs were significantly increased in the SON (+28-61%) and cortex (+27-42%) after kindling. Immunoreactivity for mGluR1 but not mGluR2/3 was significantly increased in vivo in the SON. Receptor protein expression and intracellular calcium accumulation in response to the mGluR agonist, 1S,3R ACPD, were evaluated after in vitro "kindling" of neuroendocrine cells by Mg2+ deprivation. Increased immunoreactivity for mGluR1 and mGluR2/3 was seen in all cultures 3 days after a brief exposure to Mg2+-free medium. 1S,3R 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) induced rapid peak responses and gradual accumulations of intracellular Ca2+ in neurons. Both responses were increased in the "kindled" cells. Increases in the expression of functional mGluR1 and perhaps mGluR3 receptors may contribute to the development of long-lasting plastic changes associated with seizure activity.

Channel properties of NMDA receptors on magnocellular neuroendocrine cells cultured from the rat supraoptic nucleus.

Application of N-methyl-D-aspartate (NMDA) to the supraoptic nucleus of the hypothalamus (SON) generates clustered firing that may be important in hormone release. However, synaptically evoked EPSPs recorded from SON neurons exhibit varying contributions from NMDA receptors. We used the high resolution of single-channel recording to examine the receptor and ion channel properties of NMDA receptors expressed by SON neurons in 'punch' culture. Biocytin introduced into individual neurons during patch clamp recording revealed large (32.1+/-3.3 micron), oblong somas and bipolar extensions typical of magnocellular neuroendocrine cells (MNCs). Rapid application of NMDA (100-300 microM) in the presence of 10 microM glycine to outside-out macropatches resulted in openings with an average conductance of 46. 9 pS and reversal potential of +3.9 mV. Increasing glycine from 0.03 to 30 microM increased the apparent frequency, duration and occurrence of overlapping NMDA-elicited openings. NMDA responses were inhibited by Mg2+ in a voltage-dependent manner and by the NMDA-site antagonist, D-(-)-2-amino-5-phosphonovaleric acid (D-APV). Application of saturating NMDA or glycine alone with the glycine-site antagonist, 5,7-dichlorokynurenate (DCK) or with D-APV, respectively, did not result in agonist-induced openings. NR1 immunoreactivity was observed in large neurons (>25 micron) with MNC-like morphology. These single-channel and immunocytochemical data confirm the presence of functional NR1-containing NMDA receptors in MNCs.

Cortical cell loss in asymptomatic cats experimentally infected with feline immunodeficiency virus.

Specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) were used to evaluate the development of central nervous system changes during the asymptomatic stages of viral infection. The brains of asyptomatic cats were examined at postinoculation times ranging from 8 weeks to 3 years for changes in neuron density, glutamate receptor density, and synaptophysin immunoreactivity. At 2-3 years postinoculation a small decrease in neuronal density was found in layers 2-3 and layer 5 of the frontal cortex (-14.4%), parietal cortex (-18.1%), and striatum (-29.5%). The only other indications of pathology within these regions were a mild diffuse astrogliosis, occasional microglial nodules, and the accumulation of satellite cells around selected neurons. An average loss of large neurons of 56-68% was seen in the cortex of four random source cats euthanized with AIDS. These values contrasted with the absence of any significant cell loss in FIV-infected cats 18 weeks after inoculation or FIV-negative controls. The loss of neurons in the asymptomatic cats showed a significant positive correlation with a decrease in the blood CD4:CD8 ratios. Morphometric evaluation of synaptic terminal densities immunocytochemically stained with synaptophysin revealed a significant increase in the asymptomatic cats at 2-3 years postinoculation that correlated negatively with the CD4:CD8 ratios. Random source AIDS cats showed a 34% decrease in synaptophysin-immunoreactive profiles. Glutamate binding in the cortex did not change significantly in the asymptomatic cats (4-7% decline). Thus, experimentally infected specific pathogen-free cats show a loss of cortical neurons similar to what has been observed in postmortem studies of humans infected with HIV. The detection of neuronal loss during the asymptomatic stage of disease and the correlation with the peripheral CD4:CD8 cell ratios indicate that neurodegeneration may progress in parallel with peripheral disease.

Amygdala kindling alters N-methyl-D-aspartate receptor subunit messenger RNA expression in the rat supraoptic nucleus.

Intense electrical activity throughout the brain which results from generalized epileptic or kindled seizures is thought to cause persistent and widespread neuronal plastic changes. We have previously reported that stage 5 kindled seizures cause an increase in vasopressin messenger RNA content and nitric oxide synthase activity in neuroendocrine cells of the supraoptic nucleus which lasts for at least four months after the last seizure. To evaluate whether changes in the expression of N-methyl-D-aspartate receptor subunits might contribute to these effects, the expression of NR1, NR2A, NR2B. NR2C and NR2D subunit messenger RNAs was examined by in situ hybridization in neuroendocrine cells of the supraoptic nucleus one month after amygdala kindling to stage 5 seizures. No change in NR1 subunit messenger RNA expression was seen. In contrast, NR2B subunit messenger RNA was significantly increased. by about 63%, and NR2D subunit messenger RNA was significantly decreased, by about 22%. indicating a shift in NR2 subunit messenger RNA expression. NR2B subunit messenger RNA was also significantly increased in adjacent limbic structures. The long-lasting shift towards increased NR2B and decreased NR2D messenger RNA expression after kindling suggests that N-methyl-D-aspartate receptor NR2 composition may be an important factor in the maintenance of pathological plasticity following generalized seizures. If these changes in messenger RNA are translated into increased NR2B and decreased NR2D subunits in the N-methyl-D-aspartate receptors in vivo, both a decrease in sensitivity due to a strong magnesium block and an increase in channel ion gating might be predicted.

Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in vasopressin and oxytocin neuroendocrine cells.

Vasopressin and oxytocin neuroendocrine cells within the supraoptic nucleus display distinctive electrophysiological properties and differential responses to selected NMDA receptor (NR) antagonists. To determine if these differences might be due to NMDA receptor composition, we compared the expression of NR1, NR2A, NR2B, NR2C and NR2D subunit mRNAs in immunocytochemically identified vasopressin and oxytocin neuroendocrine cells. In contrast to NR1 subunit mRNA which was equally expressed in both vasopressin and oxytocin cells, NR2B and NR2C displayed very different expression patterns. In oxytocin cells, the NR2B subunit comprised the majority (65%) of the total NR2 expression with NR2C and NR2D contributing 6% and 27%, respectively. Vasopressin cells exhibited 5-fold higher NR2C (32%), approximately half as much NR2B mRNA (39%) and equivalent NR2D (31%). In vitro expression studies have shown that the NR1-NR2C subunit combination exhibits weaker magnesium block and higher affinity for glycine than NR1-NR2B. Thus, the high expression of NR2C in vasopressin cells relative to oxytocin cells may make these cells more susceptible to glutamatergic activation. These observations in vasopressin and oxytocin cells provide the basis for a working model to investigate how differential NMDA receptor composition may shape the neurophysiological properties of neurons.

Kindling induces a long-lasting increase in brain nitric oxide synthase activity.

Generalized seizures induced by kindling are associated with a long-term increase in vasopressin mRNA expression in vasopressin neuroendocrine cells. Since nitric oxide synthase activity is strongly expressed in these neurons and may play a role in mechanisms of plasticity, we used NADPH-diaphorase histochemistry to examine nitric oxide synthase activity 1 month after amygdala kindling. Both the number of stained neurons and average intensity of cellular labeling in the supraoptic nucleus were increased in the kindled rats. In adjacent limbic regions, terminal-like staining was also increased, suggesting a general elevation of limbic nitric oxide synthase activity. Thus, increased nitric oxide production may play a role in sustaining the increase in vasopressin mRNA and plastic changes in the amygdala associated with kindling.

Increased hypothalamic glutamate receptors induced by water deprivation.

To examine the role of receptor changes in the adaptive response to physiological stimulation, the density and distribution of excitatory amino acid receptors within the hypothalamus and other brain regions were examined in rats deprived of water for 2 days. Membrane binding assay revealed an increase in glutamate receptor density and a small shift in the affinity of glutamate for the receptor. Regional analysis of these changes by receptor autoradiography specific for NMDA, non-NMDA or metabotropic glutamate receptor binding indicated that NMDA and metabotropic receptor densities are increased in the brain. Regional increases were found principally for the NMDA receptor binding within the supraoptic nucleus, anterior hypothalamus, caudate-putamen and globus pallidus with no significant changes in 24 other brain regions. No significant changes were found in any brain regions for AMPA receptors. Metabotropic and kainate receptors tended to parallel the NMDA receptor changes, although few regions reached statistical significance. These changes indicate that brain regions associated with water balance regulation show selective adaptive increases in NMDA receptors during water deprivation which may facilitate prolonged activation of these cells.

Vasopressin mRNA changes during kindling: the effects of kindling site and stage.

Because of the many anatomical and functional links to the limbic system, the neuroendocrine system is often affected by limbic disturbances. Limbic seizures in humans and animals alter neuroendocrine function and hormone levels. We have shown that in an animal model for partial seizures, the amygdala kindled rat, plasma vasopressin levels are elevated and a sustained increase in vasopressin (VP) mRNA follows stage 5 kindled seizures. In the present experiments we sought to determine when during the course of amygdala kindling the VP mRNA increase occurs and whether specific anatomical pathways mediate this increase. Animals kindled to early seizure stages (stages 1, 2 or 3) had no consistent increase in VP mRNA in the supraoptic nucleus (SON) while animals kindled to generalized seizures, stages 4 or 5, invariably had increased VP mRNA relative to controls. Electrical kindling to stage 5 seizures from two other brain sites, the dorsal hippocampus and the anterior olfactory nucleus, consistently resulted in a significant increase in VP mRNA one week after completing kindling. In all experiments the increase in VP mRNA in the SON showed no differences related to the side or proximity of the electrodes used for kindling. Measures of water balance did not change following kindling. These results indicate that kindled seizure generalization is a prerequisite for the long-term increase in VP mRNA. Furthermore, the VP mRNA increase appears to involve polysynaptic pathways accessible from different limbic kindling sites. These studies support the hypothesis that changes in mRNA regulation may contribute to the neuroendocrine pathophysiology accompanying limbic seizures.