Estrogen modulation of the cyclic AMP response element-binding protein pathway. Effects of long-term and acute treatments.

Actions of estrogen include mechanisms leading to alterations in gene transcription that may be independent of nuclear estrogen receptors, as well as those involving direct action of the estrogen receptor on the genome. Also, the influence of estrogen in the brain appears to extend well beyond areas associated with reproduction and may include forebrain areas linked to affective and cognitive behaviors. We investigated the effects of acute and long-term estradiol benzoate (E2) treatment on total and phosphorylated cyclic AMP responsive element-binding (CREB) protein levels and on cyclic AMP response element (CRE)-DNA binding in forebrain areas of ovariectomized (OVX) rats. Long-term E2 treatment increased CRE-DNA binding in the amygdala but not in hippocampus, frontal cortex, or cerebellum. The increase in CRE-DNA binding in the amygdala was associated with increased levels of total and phosphorylated CREB (pCREB) protein during protracted E2 exposure. To localize the estrogenic effect in the amygdala and determine if an effect in one hippocampal region was masked by a lack of effect in another subregion, we performed immunolabeling of pCREB in brain structures of chronically treated OVX animals with or without E2. This treatment resulted in a significant increase in relative total immunolabeled nuclei in the anteroventral subdivision of the medial amygdala. In the hippocampus, a significant increase in relative total immunolabeled nuclei was seen in the CA1 and CA3 regions, but not in the dentate gyrus or hilus of the dentate gyrus. Acute E2 treatment resulted in increased CRE-DNA binding in the frontal cortex but not in amygdala, hippocampus, or cerebellum. However, no changes in levels of total CREB or pCREB protein were observed in the frontal cortex under E2 treatment. No changes were observed either in basal or cAMP-stimulated protein kinase A (PKA) activity or in PKA-alpha catalytic subunit immunoreactivity in the amygdala or the frontal cortex. Our study indicates that both long-term and acute treatments with estrogens influence the function of CREB in specific brain structures.

Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration.

Previously, we observed that injection of an adenoviral (Ad) vector expressing glial cell line-derived neurotrophic factor (GDNF) into the striatum, but not the substantia nigra (SN), prior to a partial 6-OHDA lesion protects dopaminergic (DA) neuronal function and prevents the development of behavioral impairment in the aged rat. This suggests that striatal injection of AdGDNF maintains nigrostriatal function either by protecting DA terminals or by stimulating axonal sprouting to the denervated striatum. To distinguish between these possible mechanisms, the present study examines the effect of GDNF gene delivery on molecular markers of DA terminals and neuronal sprouting in the aged (20 month) rat brain. AdGDNF or a control vector coding for beta-galactosidase (AdLacZ) was injected unilaterally into either the striatum or the SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the side of vector injection. Two weeks postlesion, rats injected with AdGDNF into either the striatum or the SN exhibited a reduction in the area of striatal denervation and increased binding of the DA transporter ligand [(125)I]IPCIT in the lesioned striatum compared to control animals. Furthermore, injections of AdGDNF into the striatum, but not the SN, increased levels of tyrosine hydroxylase mRNA in lesioned DA neurons in the SN and prevented the development of amphetamine-induced rotational asymmetry. In contrast, the level of T1 alpha-tubulin mRNA, a marker of neuronal sprouting, was not increased in lesioned DA neurons in the SN following injection of AdGDNF either into the striatum or into the SN. These results suggest that GDNF gene delivery prior to a partial lesion ameliorates damage caused by 6-OHDA in aged rats by inhibiting the degeneration of DA terminals rather than by inducing sprouting of nigrostriatal axons.

Comparison of tyrosine hydroxylase and preproenkephalin expression in rat adrenal medullary explants in vitro and transplanted into subarachnoid space.

When adrenal medullary cells are cultured in vitro, tyrosine hydroxylase (TH) mRNA, preproenkephalin (PPEnk) mRNA, and methionine enkephalin (Mek) immunoreactivity was markedly increased compared with intact adrenal medullary cells in situ, suggesting an increased biosynthesis of catecholamines and enkephalin-containing peptides. In transplanted adrenal medullary cells in vivo, TH mRNA and TH immunoreactivity are still apparent for at least 1 year after transplantation, indicating continued capacity for catecholamine biosynthesis. PPEnk mRNA levels in surviving adrenal medullary grafted cells increased, particularly in the first week after transplantation, and remained above levels found in the intact adrenal gland for at least 1 year after transplantation. These results support other studies in our laboratory, suggesting that adrenal medullary transplants reduce pain by synthesis and secretion of both catecholamines and enkephalin-containing peptides. The differences in expression of TH mRNA and PPEnk mRNA in the adrenal medulla in situ, in explants in culture and in transplants in the spinal subarachnoid space, indicate that the mechanisms regulating the expression of neurohumoral factors depend upon environmental factors extrinsic to the medullary cells themselves.

Estrogen alters behavior and forebrain c-fos expression in ovariectomized rats subjected to the forced swim test.

Estrogen has been implicated in brain functions related to affective state, including hormone-related affective disorders in women. Although some reports suggest that estrogen appears to decrease vulnerability to affective disorders in certain cases, the mechanisms involved are unknown. We used the forced swim test (FST), a paradigm used to test the efficacy of antidepressants, and addressed the hypotheses that estrogen alters behavior of ovariectomized rats in the FST and the FST-induced expression of c-fos, a marker for neuronal activity, in the rat forebrain. The behaviors displayed included struggling, swimming, and immobility. One hour after the beginning of the test on day 2, the animals were perfused, and the brains were processed for c-fos immunocytochemistry. On day 1, the estradiol benzoate-treated animals spent significantly less time struggling and virtually no time in immobility and spent most of the time swimming. Control rats spent significantly more time struggling or being immobile during a comparable period. On day 2, similar behavioral patterns with still more pronounced differences were observed between estradiol benzoate and ovariectomized control groups in struggling, immobility, and swimming. Analysis of the mean number of c-fos immunoreactive cell nuclei showed a significant reduction in the estradiol benzoate versus control groups in areas of the forebrain relating to sensory, contextual, and integrative processing. Our results suggest that estrogen-induced neurochemical changes in forebrain neurons may translate into an altered behavioral output in the affective domain.

Regulation of neuronal nitric oxide synthase mRNA in lordosis-relevant neurons of the ventromedial hypothalamus following short-term estrogen treatment.

Short-term estrogenic regulation of neuronal nitric oxide synthase (nNOS) mRNA in the ventrolateral subdivision of the ventromedial nucleus (VLVMN), an area central to lordosis, was demonstrated using in situ hybridization. Estrogen-treated animals showed a significantly greater signal in the VLVMN, but not the arcuate or supraoptic nuclei, compared to ovariectomized controls. Neuronal NOS may be involved in early actions of estrogen in the VLVMN.

Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: evidence using in situ hybridization combined with immunocytochemistry for serotonin.

1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactive in situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5 +/- 0.86, mean +/- 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Using in situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.

Differential effects of the intraventricular administration of 6-hydroxydopamine on the induction of type II beta-tubulin and tyrosine hydroxylase mRNA in the locus coeruleus of the aging Fischer 344 rat.

Noradrenergic neurons of the locus coeruleus have been shown to respond to injury by increasing the synthesis of neurotransmitter (via the activation and induction of tyrosine hydroxylase, the rate-limiting catalyst in the production of catecholamines) and initiating compensatory axonal sprouting. However, this laboratory has recently described a significant deficit in the activation of tyrosine hydroxylase in the aged Fischer 344 rat, in contrast to the young and mature rat, following partial damage to cortical and hippocampal noradrenergic terminals induced by the neurotoxin 6-hydroxydopamine. To extend these observations, we measured changes in the relative levels of neuron-specific type II beta-tubulin and tyrosine hydroxylase mRNA in locus coeruleus neurons of 2, 12, and 24-month-old Fischer 344 rats following intraventricular infusions of 6-hydroxydopamine by using in situ hybridization histochemistry. These measures were used as markers of the responsiveness of these neurons to injury. 6-Hydroxydopamine treatment induced a persistent increase (at least 10 days) in the expression of type II beta-tubulin mRNA only in 2-month-old animals; this marker decreased in the 12 and 24-month-old animals. Relative levels of tyrosine hydroxylase mRNA increased in 2 and 12-month-old lesioned animals both 3 and 10 days post-treatment. In contrast, the induction of tyrosine hydroxylase mRNA in 24-month-old animals, seen three days post-treatment, was attenuated by 10 days. These data indicate that the capacity of locus coeruleus neurons to compensate for injury by either initiating a potential sprouting response or increasing their capacity to synthesize neurotransmitter is reduced in older animals.

Determination and cellular localization of adenylyl cyclase isozymes expressed in embryonic chick heart.

Mammalian heart has been reported to express AC isozymes (types V and VI) that are inhibited by < microM [Ca2+]; avian heart has been reported to express adenylyl cyclase activity that is inhibited by < microM [Ca2]. We have used reverse transcription polymerase chain reaction (RT-PCR) to determine that type V and VI AC mRNAs are present in freshly isolated ventricular myocytes. Subsequent RNase protection assays revealed that that the type V signal is 4-5 times that for the type VI isozyme. In situ hybridization with high specific activity cRNA probes combined with immunocytochemistry with a chick anti-myosin antibody was used to probe the cellular origins of type V and type VI AC signals. These studies show that myocytes contain messages for both the type V and VI isozymes but that AC V is the major isoform. Interestingly, while the type V AC mRNA appears to be localized primarily, if not exclusively, in myocytes, the signal for type AC VI mRNA in non-myocytes is stronger than in myocytes.

Modulation of NMDA receptor expression in the rat spinal cord by peripheral nerve injury and adrenal medullary grafting.

Excessive activation of N-methyl-D-aspartate (NMDA) receptors in the spinal cord consequent to peripheral injury has been implicated in the initiation of neuropathologic events leading to a state of chronic hyperexcitability and persistence of exaggerated sensory processing. In other CNS disease or injury states, NMDA-mediated neurotoxic damage is associated with a loss of NMDA receptors, and outcome may be improved by agents reducing NMDA activation. Previous findings in our laboratory have demonstrated that the transplantation of adrenal medullary tissue into the spinal subarachnoid space can alleviate sensory abnormalities and reduce the induction of a putative nitric oxide synthase consequent to peripheral nerve injury. In order to determine changes in NMDA receptor expression in the spinal cord following peripheral nerve injury and adrenal medullary grafting, NMDA receptor binding using a high-affinity competitive NMDA receptor antagonist, CGP-39653, and NMDAR1 subunit distribution using immunocytochemistry were investigated. Two weeks following peripheral nerve injury by loose ligation of the right sciatic nerve, either adrenal medullary or striated muscle (control) tissue pieces were implanted in the spinal subarachnoid space. Binding studies revealed a marked reduction in [3H]CGP-39653 binding at L4-L5 levels ipsilateral to peripheral nerve injury in control transplanted animals. In contrast, NMDA binding was normalized in adrenal medullary grafted animals. In addition, NMDAR1 immunoreactivity was reduced in both the dorsal horn neuropil and motor neurons of the ventral horn in animals with peripheral nerve injury, while levels in adrenal medullary grafted animals appeared similar to intact controls. These results suggest that adrenal medullary transplants reduce abnormal sensory processing resulting from peripheral injury by intervening in the spinal NMDA-excitotoxicity cascade.

Deficits in the activation and phosphorylation of hippocampal tyrosine hydroxylase in the aged Fischer 344 rat following intraventricular administration of 6-hydroxydopamine.

Tyrosine hydroxylase activity was measured under optimal and suboptimal assay conditions in hippocampal extracts from young (2 month), mature (12 month), and old (24 month) Fischer 344 male rats 72 h after the infusion of 200 micrograms of the neurotoxin 6-hydroxydopamine or vehicle into the lateral ventricle. The lesion resulted in a 45-55% decrease of tyrosine hydroxylase activity measured under optimal conditions (pH 6.1, 3.0 mM 6-methyl-5,6,7,8-tetrahydropterin) and an approximately 35% decrease in the relative concentration of immunoreactive tyrosine hydroxylase. When measured under suboptimal conditions (pH 6.6, 0.7 mM 6-methyl-5,6,7,8-tetrahydropterin), tyrosine hydroxylase activity in 2- and 12-month-old lesioned animals was twice that measured in vehicle-treated animals. However, in the old lesioned animals, tyrosine hydroxylase activity measured under suboptimal conditions was not different from that measured in age-matched vehicle-treated animals. Isoforms of tyrosine hydroxylase were identified on immunoblots after two-dimensional gel electrophoresis using enhanced chemiluminescence. The relative proportion of lower pI isoforms of tyrosine hydroxylase in the 2-month-old lesioned animals was greater than that observed in vehicle-treated controls. In contrast, no difference was seen in the relative proportion of tyrosine hydroxylase isoforms in the 24-month-old lesioned versus control animals. These data indicate that the ability of locus ceruleus neurons to rapidly respond to and compensate for insult is attenuated in 24-month-old Fischer 344 rats due to a deficit in stimulus-evoked enzyme phosphorylation.

Quantitative light microscopic localization of [3H]naltrindole binding sites in the rat brain.

The binding of radiolabeled naltrindole ([3H]NTI), a selective delta-opioid antagonist, was characterized using receptor autoradiography. Receptor binding properties were established in brain paste slices which demonstrated one site receptor occupancy with an apparent Kd of 0.25 +/- 0.08 nM (Bmax of 597.5 fmol/mg protein). Autoradiographic localization of [3H]NTI binding sites in the rat brain revealed high densities of these sites in the cortex (layers 1-3 and 6), caudate putamen, accumbens, claustrum, and internal plexiform layer of the olfactory bulb. Moderate to low levels of specific binding were observed in the hippocampus, thalamus, and substantia gelatinosa of the spinal cord.

The expression of CNTF message and immunoreactivity in the central and peripheral nervous system of the rat.

We have examined the temporal and spatial expression of ciliary neurotrophic factor (CNTF) and its messenger RNA (mRNA) by immunocytochemistry and Northern blot analysis. Specific CNTF-like (CNTF-IR) immunoreactivity and CNTF message were detected primarily in sciatic nerve, spinal cord, and optic nerve sections in the adult rat. In the sciatic nerve immunostaining was localized primarily to the Schwann cell cytoplasm. Schwann cells in the dorsal root ganglion and in the spinal motor roots were immunoreactive for CNTF but no CNTF immunoreactivity was detected in Schwann cells associated with unmyelinated sympathetic fibers in the superior cervical ganglion. CNTF-IR was first detected in sciatic nerve sections on postnatal day 8 and increased in intensity until reaching adult levels by postnatal day 21. In the adult rat, optic nerve staining was confined to astrocyte-like cells and their projections, and this pattern of immunoreactivity was first apparent at postnatal day 8. Double labelling experiments suggest that a GFAP-positive cell in the optic nerve synthesizes CNTF. In the spinal cord, CNTF-IR was seen only in white matter non-neuronal cells at all ages studied. Branched oligodendrocyte-like cells appeared stained and the staining was first apparent at postnatal day 8. The data, therefore, suggest that myelinating Schwann cells produce CNTF and demonstrate the presence of CNTF and its messenger RNA at the appropriate time period in tissues previously defined as having biological responses to CNTF.

Binding of [3H]-p-aminoclonidine to alpha 2-adrenoceptor states plus a non-adrenergic site on human platelet plasma membranes.

Characterization of the binding of [3H]p-aminoclonidine ([3H]PAC) to purified plasma membranes from human platelets has revealed multiple binding sites. [3H]PAC identified site-1 in the picomolar affinity range (site-1 KD estimates ranged from 13 to 94 pM). Site-1 displayed a rank order of competition by various compounds for [3H]PAC, indicative of an alpha 2-adrenoceptor, and was sensitive to 0.1 mM GTP. [3H]PAC also identified a second site with nanomolar affinity (site-2 KD estimates ranged from 0.7 to 1.7 nM). In the presence of 0.1 mM GTP, site-2 was not diminished significantly. Also in contrast to site-1, site-2 displayed low affinity for yohimbine (YOH), (-)-epinephrine and (-)-norepinephrine (NE). Therefore, site-2 could not be an active alpha 2-adrenoceptor; instead it had properties similar to a previously reported imidazoline-preferring binding site. A third site (site-3) bound [3H]PAC with a KD for site-3 of 26.6 +/- 10.0 nM (SD). Site-3 had a rank order of competition by various compounds for 5 nM [3H]yohimbine ([3H]YOH) binding which was indicative of an alpha 2-adrenoceptor. (-)-NE competed for 5 nM [3H]YOH binding at two sites: site-1 Ki = 32 pM, site-3 Ki = 239 nM. Treatment with 0.1 mM GTP completely removed site-1 and transferred the competitive binding of (-)-NE to low affinity (Ki = 437 nM). Thus, site-3 appears to be a free alpha 2-adrenoceptor. Bmax estimates for untreated membranes, derived from simultaneous multi-experiment curve-fitting analyses, were site-1 = 36 +/- 29 fmol/mg plasma membrane protein, site-2 = 95 +/- 34 fmol/mg and site-3 = 154 +/- 35 fmol/mg. We are the first to report a site for [3H]PAC binding on platelets (site-2) with properties uncharacteristic of an adrenoceptor. This observation appears to be due to our use of purified plasma membrane and low ionic strength buffer. These studies relate to reports of increased binding of [3H]PAC to platelets from depressed patients.

Expression of acidic fibroblast growth factor mRNA in the developing and adult rat brain.

We have localized acidic fibroblast growth factor (aFGF) mRNA in the developing and adult rat brain using in situ hybridization histochemistry. Prenatally, hybridization to aFGF mRNA was observed throughout the brain, with the strongest signal associated with cells of the developing cortical plate. Postnatally, labeling was localized to specific neuronal populations. In the hippocampus, labeling of the pyramidal cell layer and dentate granule cells was observed and became progressively more intense with maturation. Labeling was also observed in both the external and internal granule cell layers of the developing cerebellum. Pyramidal cells of the neocortex as well as neurons of the substantia nigra and locus ceruleus also express aFGF. This pattern persists into adulthood, although the intensity of the labeling is significantly reduced in the adult brain. These patterns of hybridization correlate with specific developmental events and suggest that aFGF plays a significant role in both central nervous system development and neuronal viability in the adult brain.

Hippocampal adenosine A1 receptors are decreased in Alzheimer's disease.

Previous studies showed that adenosine receptors of the temporal and frontal cortices were not affected in Alzheimer's disease (AD). Here, we assessed the specific binding of [3H]cyclohexyladenosine to adenosine1 (A1) receptors in hippocampus from AD subjects and age-matched controls. By both particulate membrane and in vitro autoradiographic receptor binding methods we demonstrate that A1 receptors are significantly reduced by 40-60% in AD subjects. Scatchard analysis showed that maximum binding capacity (Bmax) was affected and there was no evidence for a change in the affinity of the receptor for the ligand (Kd). Receptor autoradiography revealed that although several regions including CA1, CA3 and deep layer of the subiculum were affected, the loss in A1 receptors was most prominent in the molecular layer of the dentate gyrus. In view of previous evidence indicating that these receptors are associated with the perforant pathway and dendritic fields of the CA1 and CA3 regions, our findings suggest loss of the presynaptic A1 receptors on axon terminals of extrinsic pathways including the perforant path and intrinsic pyramidal neurons which release glutamate.

Identification of a subpopulation of neuropeptide Y-containing locus coeruleus neurons that project to the entorhinal cortex.

A fundamental question important to the understanding of the neurochemical organization of the central nervous system focuses on the relationships between the differential phenotypic expression of multiple neurotransmitter markers in individual neuronal populations and the factors that regulate their expression. The first approach in studying this phenomenon is the determination of specific relationships between neurochemically distinct neuronal subpopulations and their efferent targets. The pontine nucleus locus coeruleus (LC) provides a useful model for addressing this question since the projections of LC neurons are topographically organized and several neuropeptides are expressed along with noradrenergic markers in subsets of these neurons. In these studies, we have focused on defining the efferent targets of LC neurons that contain neuropeptide tyrosine (NPY)-like immunoreactivity. This has been accomplished by injecting the retrograde fluorescent tracer fluorogold into specific cortical and hippocampal targets in adult rats and identifying the proportion of retrogradely labeled LC neurons that are positive for NPY-like immunoreactivity. In agreement with other investigators, no preferential cortical projections of NPY-positive LC neurons were observed. However, when fluorogold injections included or were limited to the entorhinal cortex, a discrete cluster of round or ovoid neurons in the dorsomedial portion of the LC approximately 9.8 mm posterior to bregma were found to contain NPY-like immunoreactivity. This observation demonstrates that some topographic organization of NPY-containing LC neurons does exist. In fact, these data indicate that morphologic and topographic organization exists even within neurochemically distinct subsets of neuronal populations.

Adrenergic receptors in aging and Alzheimer's disease: increased beta 2-receptors in prefrontal cortex and hippocampus.

Loss of pigmented noradrenergic locus ceruleus neurons occurs in Alzheimer's disease (AD) and, to a lesser extent, in aging. We studied beta-adrenergic receptors and their subtypes, beta 1 and beta 2, by the specific binding of 125I-pindolol to particulate membrane preparations from prefrontal cortex, hippocampus, putamen, and cerebellum and to sections from frontal cortex by in vitro autoradiography. In prefrontal cortex from controls, numbers of total beta- and beta 2-adrenoceptors did not significantly correlate with age, but number of beta 1-adrenoceptors showed a weak but significant negative correlation. Binding in tissue particulate preparations to total beta-receptors did not reveal significant differences in samples from prefrontal cortex between AD subjects and age-matched controls. However, beta 1-adrenoceptors were decreased and beta 2-adrenoceptors were increased in number by approximately 30-50% in AD subjects. Thus, the relative ratio of beta 1-/beta 2-receptors was decreased in AD. Binding by in vitro receptor autoradiography performed in a subset of samples of frontal cortex also showed beta 2-adrenoceptors, and less consistently total beta- and beta 1-receptors, to be increased significantly in number in cortical laminae II, III, IV, and V of tissue sections from AD subjects. In these subjects, number of locus ceruleus cells and norepinephrine concentrations in putamen and frontal cortex were markedly reduced compared with values in controls. In the hippocampus, total beta- and both beta 2- and beta 1-adrenoceptors were increased in number in AD. In contrast, in the putamen, where beta 1-receptors predominate, total beta- and beta 1-receptors were significantly decreased in number with no consistent change in content of beta 2-receptors in AD. There were no significant changes in the cerebellum. Specific pindolol binding was not affected by interval between death and sampling of tissue at autopsy. Our results indicate selective changes in number of beta-receptors in AD. These changes in the cortex and hippocampus suggest receptor upregulation in response to noradrenergic deafferentation from the locus ceruleus or may simply reflect glial proliferation in AD.

Neurochemistry of dementia: establishing the links.

Neurochemical research in dementia needs to move beyond descriptive inventories of neurotransmitter systems affected in the specific disorders and to link to molecular studies of mechanism and clinical studies of cognition. New advances in Alzheimer's Disease (AD), Huntington's Disease (HD), and Parkinson's Disease (PD) are being guided by models of how nerve cells die in these disorders. Theories of pathophysiology which address the cellular level need to explain the selective vulnerability of neuronal populations in the different diseases. Clinically, the importance of neurochemical studies will be increased by understanding the bridges between neural and cognitive processes. Clinicians are concerned about the nosology of dementias, diagnostic tests, and more effective therapies. The value of neurochemical studies will be enhanced to the extent that they can contribute to understanding and modifying the clinical phenomenology of these disorders. In this paper, we will briefly review what is known about the neurochemistry of dementia but focus most of our attention on establishing the linkage between this level of description and the levels of description which are either "downstream" (molecular biology) or "upstream" (cognition) in terms of a reductionistic conception of understanding the disease process. We will explore how understanding neurochemistry relates to our understanding of disease mechanism and what constraints neurochemical studies place on understanding clinical aspects of disease. We will conclude by briefly discussing some of the problems with our current understanding of the neurochemistry of dementia and how we can address those problems in the future.

Neurochemistry of dementia.

New neurotransmitter system abnormalities are being described in Alzheimer's disease, Parkinson's disease, and Huntington's disease at a rapid rate. Both classic and neuropeptide systems are affected in cortical and subcortical regions. Comprehensive understanding of the pathophysiology of these disorders will require understanding the multisystem nature and shared features of these disorders.

New approaches to quantitative neuropathology: multivariate analysis of morphologic and neurochemical measures.

The excellent review by Coleman and Flood on neuropathological changes in normal aging and Alzheimer's disease highlights the need for development and application of computer-assisted image analysis to the study of neurons in these conditions. The morphological and neurochemical changes in normal and pathological aging require quantitation and statistical analysis that can be best performed with the assistance of the image and data processing capabilities of the computer. Advanced image processing systems are being developed to identify and classify neurons according to several intelligently chosen visual features, apply discriminant analysis and population statistics to this data, and correlate this information to other neurochemical measurements as well as the clinical history of the patient. Techniques such as immunocytochemistry, receptor autoradiography and in situ hybridization produce information-rich images of the distribution of proteins and nucleic acids in tissue slices that can be analyzed by this approach.