Increased Dementia Mortality in West Virginia Counties with Mountaintop Removal Mining?

Atmospheric particulate matter (PM) is elevated in areas of mountaintop removal mining (MTM), a practice that has been ongoing in some counties of West Virginia (WV) USA since the 1970s. PM inhalation has been linked to central nervous system pathophysiology, including cognitive decline and dementia. Here we compared county dementia mortality statistics in MTM vs. non-MTM WV counties over a period spanning 2001-2015. We found significantly elevated age-adjusted vascular or unspecified dementia mortality/100,000 population in WV MTM counties where, after adjusting for socioeconomic variables, dementia mortality was 15.60 (±3.14 Standard Error of the Mean (S.E.M.)) times higher than that of non-MTM counties. Further analyses with satellite imaging data revealed a highly significant positive correlation between the number of distinct mining sites vs. both mean and cumulative vascular and unspecified dementia mortality over the 15 year period. This was in contrast to finding only a weak relationship between dementia mortality rates and the overall square kilometers mined. No effect of living in an MTM county was found for the rate of Alzheimer's type dementia and possible reasons for this are considered. Based on these results, and the current literature, we hypothesize that inhalation of PM associated with MTM contributes to dementia mortality of the vascular or unspecified types. However, limitations inherent in ecological-type studies such as this, preclude definitive extrapolation to individuals in MTM-counties at this time. We hope these findings will inspire follow-up cohort and case-controlled type studies to determine if specific causative factors associated with living near MTM can be identified. Given the need for caregiving and medical support, increased dementia mortality of the magnitude seen here could, unfortunately, place great demands upon MTM county public health resources in the future.

Blunted amygdalar anti-inflammatory cytokine effector response to postnatal stress in prenatally stressed rats.

Smad1 is the downstream effector for bone morphogenetic protein, part of the anti-inflammatory cytokine family. Glucocorticoids (GCs) increase the production of anti-inflammatory cytokines to oppose the actions of pro-inflammatory cytokines. Here we used the prenatally stressed (PS) rat to see if chronic GC activation affects this protective mechanism in the amygdala. Male PS and control offspring were either left undisturbed or exposed to a 2-week regimen of intruder stress. One week later, half of these animals were further subjected to restraint stress for 3 days. Nuclear and cytoplasmic phosphorylated (p)-Smad1 were visualized by immunocytochemistry and quantified in the lateral and basolateral amygdala and in the hind limb primary somatosensory (S1HL) cortex. PS rats showed significantly greater baseline p-Smad1 per cell than controls. However, intruder stress increased p-Smad1 nuclear staining in the control rats only: no further increases in either compartment were observed in the PS group. With repeated restraint stress, attenuation of both cytoplasmic and nuclear p-Smad1 responses was significantly greater in controls. Thus, the overall p-Smad1 responsiveness of amygdala neurons of PS rats to life stressors is blunted. We hypothesize that the amygdala may play an essential role in initiating the cytokine response to stress in the adult rat brain. Basal p-Smad1 staining was unaffected by prenatal stress in the S1HL cortex but became elevated in the cytoplasm following intruder stress. The significance of this is unknown, but may point to a means by which stress can generally affect cells whose functions are unrelated to driving the sympathoadrenal system.

Morphological plasticity in the neural circuitry responsible for seasonal breeding in the ewe.

An increase in the response of GnRH neurons to estrogen negative feedback is responsible for seasonal anestrus in the ewe, but the underlying neural mechanisms remain largely unknown. Neural plasticity may play an important role because the density of synaptic input to GnRH neurons changes with seasons. Moreover, the transition from breeding to anestrous season requires thyroid hormones, which are also required for neuronal development. In the first experiment, we examined whether the decrease in synapses on GnRH neurons is critical for the transition to anestrus by comparing synaptic input in thyroidectomized and thyroid-intact controls, using electron microscopic analysis. Thyroidectomized ewes remained in the breeding season, but the number of synaptic contacts on their GnRH cells was not different from those in thyroid-intact ewes that were anestrus. The next experiment tested whether there was a seasonal change in morphology of the A15 dopaminergic neurons that mediate estrogen negative feedback during anestrus by analyzing synapsin-positive close contacts onto A15 neurons with confocal microscopy. There was a 2-fold increase in these close contacts onto dendrites of A15 neurons in anestrus and a corresponding increase in the length of A15 dendrites at this time of year. The increase in dendritic length was blocked by thyroidectomy, but this procedure did not significantly affect synaptic input to A15 neurons. These results provide initial evidence that the seasonal change in synapses on GnRH neurons is not sufficient for the transition into anestrus but that plasticity of the A15 dopaminergic neurons mediating estrogen negative feedback may contribute to this seasonal alteration.

Low-frequency hearing loss in prenatally stressed rats.

We investigated the possibility that hearing thresholds are altered in prenatally stressed rats raised in a normal auditory environment. Pregnant dams were assigned randomly to prenatally stressed and control groups. Half of the dams were subjected to the mild stressors of handling, exposure to a novel cage and saline injection at random times during lights-on daily. The hearing thresholds of young adult male offspring were assessed by recording auditory-evoked brainstem responses to 0.5, 1, 2, 4, 8, 16, 32 and 64 kHz pure tones. The resultant audiograms showed that prenatally stressed offspring had significantly higher hearing thresholds than control animals at 1, 2 and 4 kHz (t-tests, P<0.05). The threshold shifts caused by prenatal stress averaged 7.7 dB across frequencies. We conclude that prenatal stress causes low-frequency hearing loss, possibly due to increased vulnerability to noise-induced hearing loss, accelerated cochlear degeneration and/or disrupted cochlear development.

Perinatal corticosteroid effect on amygdala and hippocampus volume during brain development in the rat model.

Exposure of the fetus to corticosteroid during brain development has been suggested to cause permanent change in brain structure and has been associated with long term cognitive, behavioral and emotional impairment. We evaluated the effect of perinatal corticosteroid, at a dose similar to that which human fetuses are exposed, on cerebral cortex, corpus collosum, hippocampus, dentate gyrus and amygdala in a rat model. Rat pups were given betamethasone at day 1 (P1). Brain sections from the rat pups at postnatal day 45 (P45) were then analyzed. No differences were noted in the volumes of cerebral cortex, corpus collosum, hippocampus, dentate gyrus, or three nuclei of the amygdala compared to the control and sham groups. We concluded that a single course of betamethasone, at a comparable dose to that which the human fetus is exposed in clinical practice, had no effect on these regional brain volumes at this stage of development.

Increased morphological diversity of microglia in the activated hypothalamic supraoptic nucleus.

Microglia are the immune cells of the CNS. In the normal adult mammalian brain, the majority of these cells is quiescent and exhibits a ramified morphology. Microglia are perhaps best known for their swift transformation to an activated ameboid morphology in response to pathological insults. Here we have observed the responsiveness of these cells to events surrounding the normal activation of neurosecretory neurons in the hypothalamic supraoptic nucleus (SON), a well studied model of structural plasticity in the CNS. Neurons in the SON were activated by substituting 2% saline for drinking water. Brain sections were collected from four experimental groups [controls (C), 2 d-dehydrated (2D), 7 d-dehydrated (D7), and 7 d-dehydrated/21 d-rehydrated animals (R21)] and stained with Isolectin-B4-HRP to visualize microglial cells. Based on morphological criteria, we quantified ramified, hypertrophied, and ameboid microglia using unbiased stereological techniques. Statistical analyses showed significant increases in the number of hypertrophied microglia in the D2 and D7 groups. Moreover, there was a significant increase in the number of ameboid microglia in the D7 group. No changes were seen across conditions in the number of ramified cells, nor did we observe any significant phenotypic changes in a control area of the cingulate gyrus. Hence, increased morphological diversity of microglia was found specifically in the SON and was reversible with the cessation of stimulation. These results indicate that phenotypic plasticity of microglia may be a feature of the normal structural remodeling that accompanies neuronal activation in addition to the activation that accompanies brain pathology.

Divergent glial fibrillary acidic protein and its mRNA in the activated supraoptic nucleus.

Previous studies have shown decreased immunoreactive glial fibrillary acidic protein (GFAP) in the supraoptic nucleus (SON) when magnocellular neuroendocrine cells (MNCs) are activated by lactation or dehydration. This is thought to underlie structural plasticity of glial processes that occurs during these times. Here, we investigated how this apparent reduction in protein relates to GFAP mRNA expression in the dehydrated rat as visualized by in situ hybridization. Densitometry of silver grains in the SON revealed low levels of mRNA expression in control, 2-day dehydrated and 21-day rehydrated (R21) animals. Conversely, the SON from 7-day dehydrated (D7) subjects displayed significantly more silver grains. Thus, the pattern of GFAP mRNA expression is the inverse of what we previously observed for GFAP immunoreactivity in tissue sections of the SON. No differences in mRNA levels due to hydration state were seen in the lateral hypothalamic area, suggesting that increases in GFAP mRNA at D7 were specifically related to MNC activation. These data indicate a divergence in GFAP mRNA and protein expression in the SON.

Early emergence of increased fearful behavior in prenatally stressed rats.

We have been studying the mildly prenatally stressed (PS) rat as a potentially useful animal model of anxiety disorders. Previously we have demonstrated that there are anatomical and biochemical alterations in the amygdalas of adult PS offspring and that these offspring show increased fearful behaviors. However, human data indicate that anxiety disorders often present first in early childhood and then persist throughout adolescence and adulthood. To determine if PS rats also model this characteristic of human anxiety disorders, here we asked whether behavioral indices of increased fear would be detectable at an early age. We tested the hypotheses that young PS rats would show increased behavioral fearfulness in response to an acute stressor and that this would increase with age. A mild prenatal stressor, consisting of removal of the dam from the home cage and administration of a subcutaneous injection of 0.1 ml of 0.9% saline daily, was administered during the last week of pregnancy. Offspring were tested in the defensive-withdrawal apparatus before and after exposure to restraint stress at 25, 45 and 60 days of age. PS animals showed increased defensive-withdrawal behavior following the stressor and were more fearful following restraint when compared to controls (CON). This was significant at P45 and increased to P60. Hence, fearful behaviors in PS rats emerge prior to sexual maturation and increase in magnitude thereafter, further validating our model as a means to investigate the underpinnings of anxiety disorders.

Analysis of extinction acquisition to attenuated tones in prenatally stressed and non-stressed offspring following auditory fear conditioning.

Stimulus generalization occurs when stimuli with characteristics similar to a previously conditioned stimulus (CS) become able to evoke a previously conditioned response. Experimental data (Lissek et al., 2005) indicate that patients with post-traumatic stress disorder (PTSD), more often show stimulus generalization following fear conditioning when tested under laboratory conditions. Factors surrounding this observation may contribute to two common features of PTSD: 1) hyper-responsiveness to sensory stimuli reminiscent of those associated with the original trauma, and 2) resistance of PTSD to extinction-based therapies. Adverse early experience is considered a risk factor for the later development of PTSD and in the present experiments we hypothesized that stimulus generalization would occur in an animal model of adverse early experience, the prenatally stressed (PS) rat. Adult PS and control (CON) rats underwent extensive pre-habituation to a conditioning chamber followed by conventional auditory fear conditioning. The next day both groups began an extinction regimen where a series of quieter (attenuated), CSs were administered prior to the full 75 dB training CS. When tested in this manner, PS rats froze at significantly lower tone amplitudes than did CON offspring on the first day of extinction training. This suggests that the PS rats had stimulus-generalized the CS to lower decibel tones. In addition to this finding, we also observed that PS rats froze more often and longer during three ensuing days of extinction training to attenuated tones. Group differences vanished when PS and CON rats were extinguished under conventional conditions. Thus, it appears that the two extinction regimens differed in their aversive cue saliency for the PS vs. CON rats. Follow-up prefrontal cortex transcriptome probing suggests that cholinergic and dopaminergic alterations may be involved.

Differential expression of tenascin by astrocytes associated with the supraoptic nucleus (SON) of hydrated and dehydrated adult rats.

The present study evaluated the expression of tenascin by astrocytes in the supraoptic nucleus and associated ventral glial limitans (SON-VGL) under conditions that induce reversible changes in neuronal organization (dehydration and rehydration). Immunostaining of astroglia cultured from rat neonatal SON-VGL confirmed that these cells are capable of both expressing and secreting tenascin. Observations of immunostained tissue sections from adult rats revealed tenascin immunoreactivity primarily in the VGL and dendritic zone, subjacent to SON neuronal somata. Comparison of immunostained tissues from hydrated and dehydrated animals showed an apparent decrease in the intensity of immunostaining with dehydration. Subsequent Western blots of similar tissues confirmed the presence of the 210-220-kDa tenascin protein in the SON-VGL. SON-VGL tissues from control, dehydrated, and rehydrated rats were then studied by using SDS-PAGE and quantitative gel densitometry. A consistent decrease in tenascin concentration was observed by 6 days of dehydration that, with rehydration, reversed back toward or beyond control levels. Together, these observations indicate that SON-VGL astrocytes variably express tenascin and that this protein may play a role in adult SON plasticity.

Plasticity of astrocytes of the ventral glial limitans subjacent to the supraoptic nucleus.

We present evidence of gross morphological changes in astrocytes of the ventral glial limitans (VGL) associated with a well-known model of central nervous system (CNS) plasticity: the hypothalamic supraoptic nucleus (SON). Activity of SON magnocellular neuroendocrine cells (MNCs) was stimulated in experimental rats by substitution of 2% saline for drinking water for 2 or 9 days. Light microscopic measures revealed that a significant decrease in VGL thickness, by 34%, occurred with 9 days of stimulation. Astrocyte nuclei of 9-day dehydrated animals were also found to be 39% closer to the pial surface when compared with controls. Electron microscopy revealed a reorientation of individual astrocytes from a direction perpendicular (vertical) to the pial surface, to one parallel (horizontal) to this region. Vertically oriented astrocytes were found to be greater in the control group, by 49%, when compared with the 9-day dehydrated group, where cells were predominantly horizontal in orientation. Vertically oriented cells were further analyzed as to the direction of their vertical projections. Control, 2-day dehydrated and 9-day rehydrated animals, had more vertical cells which were oriented toward the pial surface when compared with 9-day dehydrated animals, where the relatively few vertically oriented astrocytes were significantly more likely to project toward the dendritic zone. In animals allowed to rehydrate for 9 days following a period of dehydration, these changes returned toward control levels. We conclude that astrocytes in vivo are capable of reversible gross morphological changes over a relatively short time.

Pharmacologically-distinct subsets of astroglia can be identified by their calcium response to neuroligands.

Type 1 astroglia have been reported to exhibit in excess of 20 different neuroligand receptors in vitro. The aim of this study was to determine if astroglia, like neurons, are heterogeneous with respect to their ability to respond to different neuroligand receptor agonists. Type 1 astroglia were loaded with the calcium indicator dye fura-2 and the influence of six different neuroligands on their intracellular calcium levels was examined using a video-based imaging system. Each of the six different neuroligands tested increased calcium levels in a subpopulation of glial fibrillary acidic protein immunopositive cells (astroglia). The percentage of cerebral cortical type 1 astroglia responding to a given neuroligand varied with the agonist and generally followed the order: 2-methylthio-ATP greater than phenylephrine greater than carbachol = serotonin greater than glutamate = histamine. The results also indicate that a single astroglial cell can respond to one agonist with a sustained rise in calcium levels and to an alternate agonist with oscillations in calcium levels. The pharmacological heterogeneity evident among astroglia does not appear to depend on cell proliferation or association with neurons. The results of our studies indicate that cultures of cerebral cortical type 1 astroglia are composed of distinct subsets of cells that can be distinguished by qualitative differences in their ability to respond to specific neuroligands with a rise in intracellular calcium.

Glial limitans elasticity subjacent to the supraoptic nucleus.

Two previous studies from our laboratory have indicated that the ventral glial limitans subjacent to the hypothalamic supraoptic nucleus (SON-VGL) undergoes a reversible thinning upon chronic activation of the magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus (SON). Numerous other studies have shown that MNC somata hypertrophy with activation. One aim of the current study was to understand better how SON-VGL thinning occurs. A second aim was to quantify overall changes of the MNC somata region due to cellular hypertrophy to compare relative changes in dimensions. Here, we undertook a light microscopic stereological investigation of the SON and the subjacent SON-VGL of Nissl stained material under basal and activated conditions. Astrocyte numbers in the underlying SON-VGL remained stable across hydration state as did the overall volume of the SON-VGL and dendritic zone reference area. How these data are consistent with our earlier observations of SON-VGL thinning was resolved by the finding of a highly significant, 30% increase in the mediolateral dimension of the SON-VGL in dehydrated rats. These observations fit well with previous work from our laboratory that demonstrates a reorientation of SON-VGL astrocytes, from vertical to horizontal, which occurs in the activated SON-VGL. We found a significant, approximately 54%, increase in the overall volume of the MNC region of the SON. No significant rostrocaudal lengthening of the SON was detected, although a trend was evident. All the observed changes reversed with rehydration. These data indicate that elasticity of the SON-VGL acts to accommodate the volume expansion of the MNCs and enables the SON-VGL to continue as an interface between the underlying cerebrospinal fluid in the subarachnoid space and the expanded SON above.

Dynamic neuronal-glial interactions in hypothalamus and pituitary: implications for control of hormone synthesis and release.

Various lines of evidence have suggested that astrocytes play a dynamic role in control of hormone synthesis and release from the CNS. The model system most studied has been the rat hypothalamo-neurohypophysial system, consisting chiefly of the supraoptic and paraventricular nuclei and their axonal terminals. Neurons of this system manufacture and secrete oxytocin and vasopressin. Electron microscopic studies have shown that certain physiological conditions (e.g., dehydration, lactation) produce increases in direct apposition among these neurosecretory cells, an effect due to withdrawal of glial processes from between the neurons. Neurohypophysial astrocytes (pituicytes) show dynamic interactions with the neurons at the level of the terminals, by engulfing them and interposing processes between the terminals and the basement membrane when hormone demand is low. Pituicyte processes retract from both areas when hormone demand is high, allowing the neuronal terminals direct access to the perivascular space. Recently, osmotic manipulations (in the physiological range) have shown that these changes can be produced in vitro in neurohypophysial explants without stimulated hormone release. Experiments on cultured adult rat pituicytes have revealed similar morphological changes in response to noradrenaline. These changes were reversed or blocked by propranolol. The increase in direct soma-somatic apposition (7-9 nm separation) of magnocellular neurons could produce a tonic rise in (K+)o which would increase protein synthesis and contribute to the raised excitability of these neurons. Also, the removal of interposed glia could allow the formation of gap junctions and specialised synapses which are known to occur between these neurons. These in turn may participate in producing the coordinated firing that maximizes hormone release. The interactions of pituicytes with the terminals in the neurohypophysis suggests that these astrocytes are also a part of the mechanism of control of hormone release.

Astroglia proliferate in response to oxytocin and vasopressin.

The effects of the peptides oxytocin and vasopressin on the proliferation of cultured cortical and hypothalamic astroglia were assessed by two corroborative methods. Both hemocytometer cell counts, and immunocytochemistry for bromodeoxyuridine (BrdU) incorporation and glial fibrillary acidic protein (GFAP) expression indicate that oxytocin increases the rate of proliferation of both cortical and hypothalamic astroglia. While vasopressin also had an effect on cortical cells, no conclusive evidence for vasopressin affecting proliferation of hypothalamic astroglia was found.

Immunoreactive glial fibrillary acidic protein in pituicytes of the rat neurohypophysis.

The glial fibrillary acidic protein (GFAP) has been associated with glial filaments. Electron microscopic examination of rat pituicytes in our laboratory has revealed few of these 8-9 nm filaments that are present in other astrocytes. Since the literature is inconsistent on the existence of filaments in pituicytes, we investigated the content of GFAP in these cells. Immunocytochemical methods revealed a strong positivity for GFAP in pituicytes. Furthermore, the primary antiserum dilution required for optimal staining suggests that there may be more GFAP in pituicytes than in other glial elements. The significance of immunoreactive GFAP in pituicytes is discussed in terms of possible functions and embryonic origins.

Lateral amygdaloid nucleus expansion in adult rats is associated with exposure to prenatal stress.

Anxiety disorders in humans have been associated with chronic activation of the hypothalamic-pituitary-adrenal axis and changes in the volume of the amygdala. Interest in the etiology of anxiety disorders has led us and others to investigate the effects of prenatal stress on the brain development of adult male rat offspring. Prenatally stressed rats represent a promising animal model for anxiety disorders in that they have already been characterized as having both upregulated corticotropin-releasing factor (CRF) brain biochemistry and altered, more fearful, behaviors. Consistent with this, there is now evidence that prenatal stress also has an impact on the development of CRFergic neurons in the hypothalamic paraventricular nucleus and neurogenesis in the hippocampus. At this time, little information about the impact of prenatal stress on amygdala anatomy has been presented. Here we asked whether prenatal stress also has an impact on the development of the amygdala, because this structure plays a direct role in the emotions of anxiety and fear. Stereological measures of well-defined subregions of amydgdaloid nuclei revealed significantly expanded dimensions of the lateral nucleus in prenatally stressed offspring, due, in part, to more neurons and glia. These data may have direct import for the effect of adverse early life experiences and the etiology of anxiety disorders in humans. They also imply that early experiences may not be "grown out of" with development; in fact, the opposite might be true-adverse early life experiences may set developmental events into motion in the brain that last a lifetime.

The surface density of glial fibrillary acidic protein immunopositive astrocytic processes in the rat supraoptic nucleus is reversibly altered by dehydration and rehydration.

Ultrastructural and immunohistochemical studies of the supraoptic nucleus (SON) have provided evidence that retraction and extension of astrocytic processes from between magnocellular neuroendocrine cells (MNCs) likely plays a role in the release of oxytocin, and/or vasopressin, that accompanies parturition, lactation and dehydration. The present study estimates the surface density (Sv) of glial fibrillary acidic protein (GFAP)-immunoreactivity, predominantly in astrocytic processes, in the SON of normally hydrated, dehydrated and rehydrated rats. The Sv of GFAP processes in dehydrated rats was significantly reduced compared with control levels. Rehydration returned Sv to control levels. The reversible reduction in Sv indicates that the previously observed reduction in optical density is due to a rearrangement of astrocyte processes in the SON which occurs at the same time as the selective functional activation of MNCs.

Glutathione S-transferase π-isoform is not altered in bladder tissue from smokers.

Glutathione S-transferase π (GSTπ)-isoform is a cytosolic enzyme involved in the detoxification of reactive metabolites generated from environmental pollutants and cigarette smoke. Although cigarette smoking has been implicated in the etiology of bladder cancer, no information yet exists regarding GSTπ in smokers and nonsmokers. This study was carried out to evaluate the immunohistochemical localization and to measure levels of immunoreactive GSTπ in bladder tissue from smokers and nonsmokers. Tissues from patients diagnosed with transitional cell carcinoma (TCC) were obtained from paraffin embedded blocks fixed in formalin. Bladder tissues from smokers (n = 7) and nonsmokers (n = 8), and histologically confirmed cancerous and noncancerous areas of the same patients (n = 10) were studied. The immunoreactive GSTπ was quantified by calculating its optical density with a computerized Olympus BH-2 microscope connected to a DAGE camera. Immunohistochemical staining for GSTπ appeared to be evenly distributed in the cytosol of the transitional epithelium (TE) of the noncancerous regions. In the TE from patients with advanced TCC, the staining intensity appeared to be stronger in the nuclei relative to cytoplasm, an effect that was even more pronounced in poorly differentiated cancers and in cancers with squamoid features. The immunoreactive levels of GSTπ in the superficial TE cells was approximately 1.7-fold higher compared with the rest of the TE layer (p < 0.05) in smokers and nonsmokers. No significant differences (p > 0.05) were observed between smokers and nonsmokers in either of these regions. The higher concentration of GSTπ in the TE is suggestive of the protective role of this enzyme, serving to prevent any potentially harmful xenobiotics from entering the bladder tissue. The lack of differences in the detoxifying enzymes between smokers and nonsmokers suggests that the increased susceptibility of bladder cancer in smokers is probably mediated by other mechanisms.

The evidence for astrocytes as a target for central noradrenergic activity: expression of adrenergic receptors.

Our recognition and understanding of adrenergic receptor expression by astrocytes and their cultured counterparts, astroglia, has occurred primarily over the past 2 decades. The advances in our knowledge have come about largely through the advent of new techniques with which to study neurotransmitter receptors, coupled with improvements in our ability to isolate, purify, and identify this central nervous system (CNS) cell type. The development of pharmacological tools such as second messenger assays, iodinated ligands, autoradiography, and intracellular electrophysiological recordings, paralleled that of cultured clonal cells lines of glial origin, purified astroglial primary cultures, isolations of astrocytes from adult tissues, and immunocytochemical staining for the astrocyte-specific glial fibrillary acidic protein (GFAP). As these techniques were combined and applied to the study of astrocyte pharmacology, our understanding of adrenergic receptor expression by these cells deepened. This review is an account of how these events have shaped our understanding of astrocytic adrenergic receptor expression.