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Histone deacetylases (HDACs) are part of a system that links epigenetic control of gene expression to a variety of environmental stimuli. Some HDACs, including HDAC4, shuttle between the cytoplasm and nucleus in response to physiological cues such as calcium signaling. HDAC4 mRNA is enriched in the brain, but the regional and subcellular protein expression pattern of HDAC4 is not known. Here we show that HDAC4 is more highly expressed in some brain regions than in others. HDAC4 is present in the perikaryial cytoplasm of most neurons but its nuclear localization is variable. In some areas, such as the dentate gyrus, nuclear expression is not detectable, whereas in other areas some neuronal nuclei contain HDAC4 immunoreactivity whereas others do not. In the cytoplasm, HDAC4 immunoreactivity is punctate. Some of these puncta are present in dendritic spines where the strongest immunoreactivity is associated with the postsynaptic density. These data demonstrate that the regional and subcellular distribution of HDAC4 is heterogeneous and raise the possibilities that HDAC4 acts on nonhistone substrates in dendritic spines or that it shuttles between spine and nucleus to coordinate synaptic activity with gene expression., (2009 Wiley-Liss, Inc.)

Rationale: In this study, we sought to examine individual differences in stress-induced behavioral sensitization to d-amphetamine after repeated social defeat stress. In an effort to understand what mechanisms underlie stress-induced sensitization to d-amphetamine, we examined striatal gene expression of the dopamine receptor D(2). Additionally, we investigated if repeated social defeat was associated with changes in dendritic spine density in the hippocampus, prefrontal cortex, and nucleus accumbens of rats that exhibit stress-induced sensitization., Methods: Male rats were classified into high responders (HR) and low responders (LR) based on their locomotor response to a novel environment. Then, rats were either handled as a control or defeated on four occasions by aggressive rats. Two weeks after the last defeat, animals were challenged with one of three doses of d-amphetamine and their locomotor activity was recorded., Results: Non-defeated HR rats exhibited higher locomotor activity in response to d-amphetamine when compared to LR non-defeated rats. Fourteen days from the last repeated social defeat, LR rats and HR rats were behaviorally identical in response to acute injections of amphetamine. Furthermore, HR non-defeated rats had less D(2) mRNA expression in the nucleus accumbens core and dorsal striatum than do LR non-defeated rats. However, after repeated social defeat, HR and LR rats had identical D(2) mRNA expression in both the core and dorsal striatum. Finally, there were no changes in dendritic spine density in any of the brain areas examined in LR rats., Conclusion: Repeated social defeat abolishes individual differences in behavioral responses to d-amphetamine which may be due to a down-regulation of striatal dopamine D(2) receptors in LR rats.

Dendritic spines are small protrusions that receive 90% of excitatory cortical synapses and are critically important to neural function. Each dendritic spine is supported by a dynamic actin cytoskeleton that responds to internal and external cues to allow spine development, elongation, retraction and movement. Multiple proteins have roles in spinogenesis, but until now, a regulatory role for actin itself has not been established. Here, we show that, in the acute slice preparation, actin expression increases during a period of rapid spinogenesis. Furthermore, actin overexpression in organotypic hippocampal cultures leads to a significant increase in spine density on CA1 pyramidal cells. Specifically, the number of filopodia (long, thin protrusions without heads) increases by 38% on secondary apical dendrites and 88% on basal dendrites and the number of elongated spines with heads increases by 162% on secondary apical dendrites and 113% on basal dendrites. Synapsin-I immunostaining demonstrated that the majority of filopodia and elongated spines are apposed by axon terminals. Additionally, we show that overexpressed actin enters both new and established spines within 24 h. These data demonstrate that neurons undertaking spinogenesis upregulate actin expression, that actin overexpression per se increases spine density, and that both new and established spines incorporate exogenous actin.

Spinophilin is an actin binding protein that positions protein phosphatase 1 next to its substrates in dendritic spines. It contains a single PDZ domain and has the biochemical characteristics of a cytoskeletal scaffolding protein. Previous studies suggest that spinophilin is present in most spines, but the concentration of spinophilin varies from brain region to region in a manner that does not simply reflect differences in spine density. Here, we show that spinophilin is enriched in the great majority of dendritic spines in cerebral cortex, caudatoputamen, hippocampal formation, and cerebellum, irrespective of regional differences in spinophilin concentration. In addition, spinophilin is present postsynaptic to asymmetrical contacts on interneuronal dendritic shafts. We further show that, in hippocampus and ventral pallidum, spinophilin is occasionally present in dendritic shafts adjacent to gamma-aminobutyric acid-containing contacts. Thus, the functional role of spinophilin may not be exclusively restricted to excitatory synapses and may be significant at a small fraction of inhibitory contacts. These data also suggest that the concentration of spinophilin per spine is variable and is likely regulated by local physiological factors and/or regional influences.

Dendritic spines, small protrusions from dendritic shafts, receive most of the excitatory synapses in cortical regions. Spines are highly plastic structures that can be rapidly produced or lost in response to a wide array of internal and external stimuli, and they proliferate in acute slice preparations [J. Neurosci. 19 (1999) 2876]. The goal of the present study was to determine if protein synthesis is necessary for this spine proliferation. We found that the addition of protein synthesis inhibitors to acute slices (in which spines otherwise proliferate) blocked new spine growth. Furthermore, a population of longer spines was observed after 2 h but these did not develop during protein synthesis blockade. These data suggest that protein synthesis is necessary for new spine growth in acute brain slice preparations and support literature suggesting that newly produced spines develop from filopodia-like protrusions.

Repeated exposure to cocaine produces an enduring increase in dendritic spine density in adult rat nucleus accumbens. It has been shown previously that chronic cocaine administration increases the expression of cyclin-dependent kinase-5 in this brain region and that this neuronal protein kinase regulates cocaine-induced locomotor activity. Moreover, cyclin-dependent kinase-5 has been implicated in neuronal function and synaptic plasticity. Therefore, we studied the involvement of this enzyme in cocaine's effect on dendritic spine density. Adult male rats, receiving intra-accumbens infusion of the cyclin-dependent kinase-5 inhibitor roscovitine or saline, were administered a 28-day cocaine treatment regimen. Animals were killed 24-48 h after the final cocaine injection and their brains removed and processed for Golgi-Cox impregnation. Our findings demonstrate that roscovitine attenuates cocaine-induced dendritic spine outgrowth in nucleus accumbens core and shell and such inhibition reduces spine density in nucleus accumbens shell of control animals. These data indicate that cyclin-dependent kinase-5 is involved in regulation of, as well as cocaine-induced changes in, dendritic spine density., (Copyright 2003 IBRO)

Olfactory bulbectomy, neonatal clomipramine administration, and maternal deprivation have been employed as animal models of depression. Each model is unique with respect to the experimental manipulations required to produce "depressive" signs, expression and duration of these signs, and response to antidepressant treatments. Dendritic spines represent a possible anatomical substrate for the enduring changes seen with depression and we have previously shown that chronic antidepressant drug exposure alters the density of hippocampal dendritic spines in an enduring fashion. The purpose of the present study was to determine whether persistent alteration of hippocampal spine density is a common element in each of these different models of depression and whether such alterations could be reversed with chronic antidepressant treatment. The results show that olfactory bulbectomy reduced spine density in CA1, CA3, and dentate gyrus compared to sham-operated controls. Chronic treatment with amitriptyline, a tricyclic antidepressant, reversed the bulbectomy- induced reduction in dendritic spine density in CA1, CA3, and dentate gyrus, whereas treatment with mianserin, an atypical antidepressant, reversed this reduction only in dentate gyrus. On the other hand, neither neonatal clomipramine administration nor maternal deprivation affected hippocampal dendritic spine density. Repeated neonatal handling, however, as a control or as part of the maternal deprivation procedure, elevated spine density in dentate gyrus. These data suggest that long-lasting alterations in hippocampal dendritic spine density contribute to the neural mechanism underlying the olfactory bulbectomy model of depression, but not the neonatal clomipramine or maternal deprivation models., (Copyright 2001 Wiley-Liss, Inc.)

Plasticity in dendritic spines may underlie learning and memory. Spinophilin, a protein enriched in dendritic spines, has the properties of a scaffolding protein and is believed to regulate actin cytoskeletal dynamics affecting dendritic spine morphology. It also binds protein phosphatase-1 (PP-1), an enzyme that regulates dendritic spine physiology. In this study, we tested the role of spinophilin in conditioned taste aversion learning (CTA) using transgenic spinophilin knockout mice. CTA is a form of associative learning in which an animal rejects a food that has been paired previously with a toxic effect (e.g., a sucrose solution paired with a malaise-inducing injection of lithium chloride). Acquisition and extinction of CTA was tested in spinophilin knockout and wild-type mice using taste solutions (sucrose or sodium chloride) or flavors (Kool-Aid) paired with moderate or high doses of LiCl (0.15 M, 20 or 40 mL/kg). When sucrose or NaCl solutions were paired with a moderate dose of LiCl, spinophilin knockout mice were unable to learn a CTA. At the higher dose, knockout mice acquired a CTA but extinguished more rapidly than wild-type mice. A more salient flavor stimulus (taste plus odor) revealed similar CTA learning at both doses of LiCl in both knockouts and wild types. Sensory processing in the knockouts appeared normal because knockout mice and wild-type mice expressed identical unconditioned taste preferences in two-bottle tests, and identical lying-on-belly responses to acute LiCl. We conclude that spinophilin is a candidate molecule required for normal CTA learning.

Cocaine enhances dopamine-mediated neurotransmission by blocking dopamine re-uptake at axon terminals. Most dopamine-containing nerve terminals innervate medium spiny neurons in the striatum of the brain. Cocaine addiction is thought to stem, in part, from neural adaptations that act to maintain equilibrium by countering the effects of repeated drug administration. Chronic exposure to cocaine upregulates several transcription factors that alter gene expression and which could mediate such compensatory neural and behavioural changes. One such transcription factor is DeltaFosB, a protein that persists in striatum long after the end of cocaine exposure. Here we identify cyclin-dependent kinase 5 (Cdk5) as a downstream target gene of DeltaFosB by use of DNA array analysis of striatal material from inducible transgenic mice. Overexpression of DeltaFosB, or chronic cocaine administration, raised levels of Cdk5 messenger RNA, protein, and activity in the striatum. Moreover, injection of Cdk5 inhibitors into the striatum potentiated behavioural effects of repeated cocaine administration. Our results suggest that changes in Cdk5 levels mediated by DeltaFosB, and resulting alterations in signalling involving D1 dopamine receptors, contribute to adaptive changes in the brain related to cocaine addiction.

The density of dendritic spines, the postsynaptic sites of most excitatory synapses, increases during the first 2 postnatal months in rat hippocampus. Significant alterations in hippocampal levels of serotonin and norepinephrine impact synaptic development during this time period. In the present study, dendritic spine density was studied in the hippocampus (CA1) and dentate gyrus of juvenile rats acutely and chronically exposed to antidepressant drugs that act on serotonin and norepinephrine. One group of 21-day-old rats was given a single injection of a serotonin specific re-uptake inhibitor (fluoxetine or fluvoxamine), a norepinephrine-specific re-uptake inhibitor (desipramine), or saline and killed after 24 h. A second group of rats was injected daily, beginning on postnatal day (PN) 21, for 3 weeks. This group was further subdivided into rats that were killed 1 day or 21 days after the last injection. Golgi analysis showed that a single injection of fluvoxamine produced a significant increase in dendritic spine density in stratum radiatum of CA1 and in the dentate gyrus. Further, acute treatment with all three antidepressants increased the total length of secondary dendrites in CA1, with fluoxetine and desipramine increasing the number of secondary dendrites as well. In fluoxetine-treated animals killed on days 42 or 62 (1 or 21 days post-treatment, respectively), dendritic spine density remained at levels present in CA1 at 21 days. These results show that acute antidepressant treatment can impact dendritic length and spine density, and raise the possibility that chronic fluoxetine treatment arrests spine development into young adulthood.

Spinophilin, a protein that interacts with actin and protein phosphatase-1, is highly enriched in dendritic spines. Here, through the use of spinophilin knockout mice, we provide evidence that spinophilin modulates both glutamatergic synaptic transmission and dendritic morphology. The ability of protein phosphatase-1 to regulate the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors was reduced in spinophilin knockout mice. Consistent with altered glutamatergic transmission, spinophilin-deficient mice showed reduced long-term depression and exhibited resistance to kainate-induced seizures and neuronal apoptosis. In addition, deletion of the spinophilin gene caused a marked increase in spine density during development in vivo as well as altered filopodial formation in cultured neurons. In conclusion, spinophilin appears to be required for the regulation of the properties of dendritic spines.

DARPP-32, a dopamine and cAMP-regulated phosphoprotein with an apparent molecular weight of 32 kD, is enriched in dopaminoceptive brain regions. Chief among these regions is the caudatoputamen which contains a large number of DARPP-32-containing medium sized spiny neurons. Since medium spiny neurons are a heterogeneous population with respect to connections and chemical neuroanatomy, it seemed of interest to determine whether DARPP-32 is present in all medium-sized neurons, or only within a specific subpopulation. The present study used immunocytochemistry and quantitative analysis to address this issue. We demonstrate that DARPP-32 is contained in almost all medium-sized neurons (96.4%) and is not detected in large neurons. Taken together with previous observations that the DARPP-32-containing medium-sized neurons project heavily to all neostriatal targets, these data demonstrate that DARPP-32 is present in virtually all neostriatal output neurons. Thus, the DARPP-32 cascade represents a final common pathway through which convergent afferent fibers using a variety of neurotransmitter agents may modulate striatal outflow., (Copyright 1998 Elsevier Science B.V.)

Dopaminergic neurons exert a major modulatory effect on the forebrain. Dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein (32 kilodaltons) (DARPP-32), which is enriched in all neurons that receive a dopaminergic input, is converted in response to dopamine into a potent protein phosphatase inhibitor. Mice generated to contain a targeted disruption of the DARPP-32 gene showed profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine, drugs of abuse, and antipsychotic medication. The results show that DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission.

Dendritic spines receive the vast majority of excitatory synaptic contacts in the mammalian brain and are presumed to contain machinery for the integration of various signal transduction pathways. Protein phosphatase 1 (PP1) is greatly enriched in dendritic spines and has been implicated in both the regulation of ionic conductances and long-term synaptic plasticity. The molecular mechanism whereby PP1 is localized to spines is unknown. We have now characterized a novel protein that forms a complex with the catalytic subunit of PP1 and is a potent modulator of PP1 enzymatic activity in vitro. Within the brain this protein displays a remarkably distinct localization to the heads of dendritic spines and has therefore been named spinophilin. Spinophilin has the properties expected of a scaffolding protein localized to the cell membrane and contains a single consensus sequence in PSD95/DLG/zo-1, which implies cross-linking of PP1 to transmembrane protein complexes. We propose that spinophilin represents a novel targeting subunit for PP1, which directs the enzyme to those substrates in the dendritic spine compartment, e.g., neurotransmitter receptors, which mediate the regulation of synaptic function by PP1.

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, apparent molecular weight of 32,000) is part of the D1 dopamine receptor signal transduction cascade. Both the D1 receptor and DARPP-32 are found in the caudate putamen, but it is not known if they co-localize in the medium-sized spiny neurons. In the present study, double-labelling immunocytochemistry was used to simultaneously localize the D1 receptor and DARPP-32 in the rat caudate-putamen. The neuropil was heavily and uniformly immunoreactive for both the D1 receptor and DARPP-32. All cell bodies immunopositive for the D1 receptor were immunopositive for DARPP-32. The D1 receptor was not detectable, however, in nearly half of the DARPP-32-containing cell bodies. DARPP-32 is present in striatopallidal and striatonigral projections. The D1 receptor co-localized with DARPP-32 in fibres of the entopeduncular nucleus and the pars reticulata of the substantia nigra. In the globus pallidus, however, D1 receptor immunoreactivity was barely detectable, while DARPP-32 immunolabelling of axons and axon terminals was intense. These data suggest that the striatal somata containing both the D1 receptor and DARPP-32 project to the entopeduncular nucleus and substantia nigra, whereas somata containing only DARPP-32 immunoreactivity project to the globus pallidus. Thus, the differences in expression of the D1 receptor and of DARPP-32 within striatal cell bodies are likely reflected in their projections. The co-localization of the D1 receptor and DARPP-32 is consistent with the known regulation of DARPP-32 phosphorylation by D1 receptor activation. The demonstration of a large population of striatal neurons that contain DARPP-32 but apparently do not contain D1 receptors substantiates the premise that these cells have an alternative signal transduction pathway. Subsequent studies are needed to search for a signal transduction pathway for these neurons analogous to the dopamine D1 receptor pathway.

Phosphoproteins and their associated kinases and phosphatases play a major role in second messenger signal cascades in neurons. Immunocytochemical studies have shown that some of these phosphoproteins and their associated enzymes are homogeneously distributed in brain and function in signal cascades common to all neurons. Other phosphoproteins and their associated enzymes are heterogeneously distributed and function in signal cascades related to specific neuronal subpopulations. Phosphoproteins and their kinases and phosphatases have been localized in the brain by electron microscopic immunocytochemistry, allowing their subcellular distributions to be analyzed. A sensitive technique employing avidin, biotin, and horseradish peroxidase has been especially useful in this endeavor. This method, along with suggestions for optimizing fixation and antibody incubations, is summarized.

Protein phosphatase 1 (PP1) is a highly conserved enzyme that has been implicated in diverse biological processes in the brain as well as in nonneuronal tissues. The present study used light and electron microscopic immunocytochemistry to characterize the distribution of two PP1 isoforms, PP1 alpha and PP1 gamma 1, in the rat neostriatum. Both isoforms are heterogeneously distributed in brain with the highest immunoreactivity being found in the neostriatum and hippocampal formation. Further, both isoforms are highly and specifically concentrated in dendritic spines. Weak immunoreactivity is present in dendrites, axons, and some axon terminals. Immunoreactivity for PP1 alpha is also present in the perikaryal cytoplasm and nuclei of most medium- and large-sized neostriatal neurons. The specific localization of PP1 in dendritic spines is consistent with a central role for this enzyme in signal transduction. The data support the concept that, in the course of evolution, spines developed as specialized signal transduction organelles enabling neurons to integrate diverse inputs from multiple afferent nerve terminals.

The localization of amyloid precursor protein (APP) in rat brain was studied with a cytoplasmic domain-specific antibody. Light microscopic immunocytochemistry demonstrated that APP is present in most neurons, in some oligodendrocytes, and in a population of cells with diameters less than 10 microns that may be glial. Marked differences in immunoreactivity among neurons were observed, and the strongest immunoreactivity was contained in larger neurons. Neurons with scant cytoplasm, such as granule cells in the olfactory bulb, dentate gyrus, and cerebellum, were weakly immunoreactive. Differences in neuropil immunoreactivity were also observed; this type of staining was strongest in the caudatoputamen, lateral septum, medial habenula, nucleus reticularis of the dorsal thalamus, and the lateral portion of the ventroposterior nucleus. Neuropil immunostaining was weakest in layer IV of cortex and in areas containing granule cells. The fact that APP seems to be present in the vast majority of neurons suggests that this protein plays a role common to all neurons. The fact that there is a great difference in the steady-state amount of APP among different types of neurons suggests that APP may play a specific role in the function of certain classes of neurons.

The localization of DARPP-32, a dopamine and cAMP-regulated phosphoprotein, has been studied in monkey brain by immunocytochemistry. This study indicates that DARPP-32 is enriched in neurons in regions receiving a dense dopamine input from the substantia nigra and ventral tegmental area. Thus, the majority of somata in the anterior olfactory area, nucleus accumbens, caudate nucleus, and putamen are immunoreactive for DARPP-32. In the caudate nucleus, immunoreactive spines receive asymmetric contacts from unlabeled axon terminals. Immunoreactive somata have diameters of 10-15 microns. In regions known to receive projections from these nuclei, immunoreactivity is confined to small puncta that represent axons and axon terminals. Regions in which immunoreactivity is present in puncta include the ventral pallidum, globus pallidus, and substantia nigra pars reticulata. Dopaminergic neurons themselves are not immunoreactive. Neurons containing moderate to weak immunoreactivity for DARPP-32 are observed in portions of the cerebral cortex, particularly in the temporal cortex (layer VI). DARPP-32-positive neurons are also present in the cerebellum, in the medial habenula, and in portions of the bed nucleus of the stria terminalis and amygdaloid complex. DARPP-32 immunoreactivity is also present in astrocytes in the subcortical white matter and in tanycytes in the arcuate nucleus and median eminence. DARPP-32 may be an effective marker for dopaminoceptive neurons in which the actions of dopamine on the D-1 dopamine receptor are mediated through cAMP and its associated protein kinase.

DARPP-32 immunocytochemistry and retrograde axonal labeling were combined to determine whether DARPP-32-containing neurons of the rat anterior cingulate cortex project to thalamus. Following injections of fluorescent latex microspheres into the mediodorsal thalamic nuclei, a large proportion of the DARPP-32 immunostained neurons in layer VI were also retrogradely labeled. In area 24a, these neurons were mostly found in layer VIb, whereas in area 24b, they were visible throughout layer VI. The presence of DARPP-32 in certain corticothalamic neurons suggests that these cells may be modulated by dopamine, which increases DARPP-32 phosphorylation, and possibly by glutamate, which antagonizes DARPP-32 phosphorylation via the N-methyl-D-aspartate (NMDA) receptor.

DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, has been studied by light and electron microscopical immunocytochemistry in the rat caudatoputamen, globus pallidus and substantia nigra. In the caudatoputamen, DARPP-32 was present in neurons of the medium-sized spiny type. Immunoreactivity for DARPP-32 was present in dendritic spines, dendrites, perikaryal cytoplasm, most but not all nuclei, axons and a small number of axon terminals. Immunoreactive axon terminals in the caudatoputamen formed symmetrical synapses with immunolabeled dendritic shafts or somata. Neurons having indented nuclei were never immunoreactive. In the globus pallidus and substantia nigra pars reticulata, DARPP-32 was present in myelinated and unmyelinated axons and in axon terminals. The labelled axon terminals in these regions formed symmetrical synaptic contacts on unlabelled dendritic shafts or on unlabelled somata. These data suggest that DARPP-32 is present in striatal neurons of the medium-sized spiny type and that these DARPP-32-immunoreactive neurons form symmetrical synapses on target neurons in the globus pallidus and substantia nigra. The presence of DARPP-32 in these striatal neurons and in their axon terminals suggests that DARPP-32 mediates part of the response of medium-size spiny neurons in the striatum to dopamine D-1 receptor activation.

The borders of the ventral striatum and ventral pallidum of the rat brain have been studied with immunocytochemistry for two protein markers that are present in these regions. One of these, DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein, Mr 32,000), is specifically enriched in medium-sized spiny neurons of the neostriatum and in the projection of these neurons upon pallidal regions. The second protein, synaptophysin, a marker for nerve terminals, effectively labels pallidal synapses. In the ventral striatum, neurons strongly immunoreactive for DARPP-32 were densely packed throughout its three components, i.e., the fundus striati and the nucleus accumbens septi, the olfactory tubercle, and the cell bridges that connect the tubercle with the overlying caudatoputamen and the nucleus accumbens. In the ventral pallidum, axons and axon terminals immunoreactive for DARPP-32 and axon terminals immunoreactive for synaptophysin were clearly delineated. As defined by these markers, the ventral pallidum was traced rostroventrally from the globus pallidus to the superficial layers of the olfactory tubercle, medially to the insula Calleja magna and the lateral septum, laterally to the pyriform cortex, and caudally to the anterior amygdaloid area. The ventral striatum and pallidum were densely intermingled in parts of the olfactory tubercle and medial forebrain bundle regions, and clearly separated in more caudal regions. The insulae Callejae did not contain typical striatal or pallidal staining patterns. Our results indicate that the ventral striatopallidal complex in the rat extends both rostrocaudally and dorsoventrally, in a highly complex, intermingled fashion, throughout most of the basal forebrain.

DARPP-32, a phosphoprotein enriched in dopaminoceptive brain neurons containing the D-1 receptor subtype, probably functions as an intracellular third messenger to mediate some of the physiological effects of dopamine at the D-1 receptor. By immunohistochemistry in rat, cat, Rhesus monkey, and human, we have localized DARPP-32 to the non-pigmented epithelium of the ciliary body, the innermost layer of the bi-layered epithelium responsible for secretion of aqueous humor into the eye. The immunoreactive protein in rat ciliary body, identified by immunolabeling of a ciliary body extract separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, is indistinguishable from DARPP-32 derived from rat caudatoputamen. By analogy with brain, we propose that DARPP-32 may act as a third messenger in the ciliary epithelium, probably through a dopaminergic mechanism.

Calcium/calmodulin-dependent protein kinase II (CaM kinase II) is a prominent enzyme in mammalian brain capable of phosphorylating a variety of substrate proteins. In the present investigation, the subcellular and regional distribution of CaM kinase II has been studied by light and electron microscopic immunocytochemistry using an antibody that recognizes the Mr 50,000 and 60,000/58,000 subunits of the enzyme. Light microscopy demonstrates strong immunoreactivity in neuronal somata and dendrites and weak immunoreactivity in axons. Electron microscopy, in addition to confirming light microscopic observations, reveals moderate immunoreactivity in spines and weak immunoreactivity in nerve terminals. An accumulation of immunoreaction product is also present on postsynaptic densities. The presence of CaM kinase II in diverse structures throughout the neuron supports the view that this enzyme may be involved in mediating a variety of calcium-dependent physiological processes. CaM kinase II immunoreactivity is present in neurons throughout the brain, but a marked regional variation in the strength of the immunoreactivity exists. Overall, there is a gradient of staining intensity with the strongest immunoreactivity in the telencephalon and the weakest in the myelencephalon. The most heavily labeled regions of the telencephalon are the hippocampal formation, lateral septum, cortical regions, neostriatum, and amygdaloid complex.

The fine structure and norepinephrine content of small granular vesicle-containing profiles were studied in normal and norepinephrine-depleted cerebral cortex of the turtle, Pseudemys. The cortex was fixed for electron microscopy with the KMnO4 procedure of Koda and Bloom ('77), while the norepinephrine content was assayed wit the radioenzymatic method of Coyle and Henry ('73). Green fluorescent fibers have been described by Parent and Poitras ('74) as located almost exclusively in the outer half of the molecular layer in turtle cortex. Small granular vesicle-containing profiles are found down to 100 microns below the pial surface, but over 50% lie within 20 microns of the surface. Within the outer 100 microns of cortex, the frequency of labeled varicosities is 1.39/1,000 microns2. The average area of the norepinephrine-containing varicosities is 0.61 microns2, and there is a mean of 18.4 vesicles per single section. The average number of large plus small vesicles in an entire varicosity was estimated to be 72. Synaptic membranes are not well-preserved with KMnO4 fixation, but good examples were found of small granular vesicle-containing profiles forming both symmetrical and asymmetrical membrane differentiations. Only a small percentage of the small granular vesicle profiles were associated with a synaptic membrane differentiation in single sections. When norepinephrine-fiber synapses are seen, they usually share a postsynaptic element with another unlabeled vesicle-containing profile. Normal turtle cortex contains an average norepinephrine concentration of 1.95 micrograms/gr, which is about eight times higher than in rat cortex. The ratio of norepinephrine to dopamine is about 18 to one, suggesting that dopamine is present predominantly in a precursor pool for norepinephrine. Small granular vesicle-containing profiles were eliminated after treatment with reserpine and 6-hydroxydopamine in concentrations that were shown to reduce norepinephrine concentration by 94% and 86%, respectively. The labeled varicosities were partially depleted by midbrain hemisection and by an inhibitor of dopamine-beta-hydroxylase (FLA-63). The norepinephrine-containing varicosities are remarkably coextensive with the distribution of thalamic fibers, both in the total extent of cortex where they are found and in the depth of cortex where they terminate. The results support the idea that there is a close structural and functional association between locus coeruleus and thalamic fibers in cerebral cortex, and the apparent difference in frequency of synapses suggests that each fiber system exerts its influence on cortical cells in a different way.

Three extrathalamic subcortical inputs to the part of the cerebral cortex that is known to receive thalamic fibers in the turtle were examined in the present study. Direct projections from the locus coeruleus, the superior medial raphe nucleus, and a wide area of the basal telencephalon that lies ventromedial to the globus pallidus were demonstrated with the horseradish peroxidase method. Fluorescence histochemistry confirmed the presence of catecholamine-containing fibers in the rostral half of dorsal cortex and also demonstrated a dense network of serotoninergic fibers. Biochemical analysis showed the concentration of both monoamines to be relatively high; the norepinephrine concentration was 709 ng/g and the serotonin concentration was 1,750 ng/g. No evidence was found to suggest the existence of either a dopamine fiber projection to cortex comparable to that of mammalian neocortex or the presence of an epinephrine pathway to turtle cortex equivalent to the epinephrine-containing fibers in the pallium of amphibians. The coexistence of the projections from the thalamus with noradrenergic projections from the locus coeruleus, serotoninergic projections from the superior medial raphe nucleus, and presumably cholinergic projections from the basal telencephalon provide at least four distinct subcortical inputs to the reptilian dorsal cortex. Neither thalamic nor similar extrathalamic inputs have been demonstrated in the dorsal pallium of amphibia. Mammalian neocortex, in contrast, has even more elaborately differentiated inputs of both types. These results support the idea that thalamic and extrathalamic inputs to cortex appear at the same time in vertebrate evolution, and that both types of inputs are required for the normal development and function of neocortex.

The cellular and subcellular distribution of protein I, a major brain phosphoprotein, has been studied in the peripheral nervous system. The levels of protein I in various peripheral nerves and innervated peripheral tissues were determined by radioimmunoassay and radioimmunolabeling of polyacrylamide gels. The results indicated tha protein I is present throughout the peripheral nervous system. Denervation studies of adrenal medulla and iris suggested that the protein I contained in peripheral tissues is localized to the neuronal elements innervating those tissues. Protein I was found to be enriched in neurotransmitter vesicle fractions of peripheral nervous tissue. Moreover, protein I appeared to be transported from cell bodies to axons terminals at least partly in association with neurotransmitter vesicles.

The ontogeny of a dopamine and cyclic adenosine-3':5'-monophosphate-regulated phosphoprotein with an apparent molecular weight of 32 kilodaltons (DARPP-32) has been studied in the central nervous system of the prenatal, newborn and adult mouse. DARPP-32-immunoreactive somata were first identified at day 12 of gestation, in the primary olfactory cortex and in the ventrolateral medulla oblongata. On day 14 of gestation, neurons containing DARPP-32-like immunoreactivity became apparent in the caudate nucleus, olfactory tubercle, nucleus accumbens, frontoparietal cortex and the ventral medulla oblongata. During the period up to and including birth, the number of cell bodies and fibres in all these areas increased markedly. In addition, DARPP-32-positive neurons became visible in the olfactory nucleus, the arcuate nucleus, and DARPP-32-positive cells appeared in the choroid plexus of the lateral, third and fourth ventricles. DARPP-32-containing fibres could be seen in the median eminence, the ventrolateral thalamus, and in the striatonigral projection, descending in the internal capsule to ramify extensively in the substantia nigra. Only in the cerebellum and suprachiasmatic nucleus did the development of DARPP-32-like immunoreactivity occur postnatally. The development of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis, was simultaneously examined. The arrival of the tyrosine hydroxylase-containing projection to the caudate nucleus, the olfactory tubercle and the nucleus accumbens apparently occurred 1-2 days after the appearance of DARPP-32-immunoreactive cells within these regions. In the ventral and ventrolateral medulla oblongata, and the primary olfactory cortex, no tyrosine hydroxylase innervation was seen near the DARPP-32-positive neurons at days 12-14. The organization of the DARPP-32-containing somata of the caudate nucleus into aggregates of 5-15 neurons was partly paralleled spatially by an increased density of tyrosine hydroxylase-positive fibres. Many DARPP-32-immunoreactive cells in the immature mouse brain are present by the day of birth, particularly in the areas known to receive a dopaminergic innervation. The development of these presumptive dopaminoceptive DARPP-32-containing neurons does not seem to be dependent on the presence, however, of a dopaminergic input, since in all regions examined DARPP-32-LI preceded the appearance of tyrosine hydroxylase-like immunoreactivity by at least 1-2 days. Indeed, the results suggest that the existence of DARPP-32-like immunoreactivity in cell bodies and dendrites may be a pre-requisite for the formation or subsequent stabilization of dopaminergic synapses.

Tyrosine hydroxylase-like, dopamine- and cyclic AMP-regulated phosphoprotein (Mr = 32,000)-like and enkephalin-like immunoreactive profiles and their codistribution have been evaluated at three rostrocaudal levels of the rat neostriatum by means of a computer-assisted morphometrical method, which allows an objective definition of high density/intensity patches using specific antibodies in combination with the peroxidase-antiperoxidase technique. Our results show that both tyrosine hydroxylase-like, dopamine- and cyclic AMP-regulated phosphoprotein-like and enkephalin-like profiles are organized in patches in the rat neostriatum. In the marginal zone, the tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches both occupied a large part of the total area. Moreover, in this zone, these putative markers for pre- and postsynaptic elements of dopaminergic synapses also showed a complete spatial overlap. In contrast, the enkephalin-like immunoreactive patches in the marginal zone occupied a smaller area, and showed only an incomplete, albeit significant overlap with the tyrosine hydroxylase-like immunoreactive/dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive system. In the central zone, tyrosine hydroxylase-like immunoreactive, dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive and enkephalin-like immunoreactive patches occupied a much smaller part of the total area than did those in the marginal zone. Within the central zone, enkephalin-like immunoreactive patches occupied a significantly larger area than did the tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches. No consistent pattern of overlap between the three different staining patterns could be seen in the central zone, probably due to the small, inconsistent size of the patches. Trend analysis showed a consistent trend of more tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches in the dorsal than in the ventral striatum, and a trend of more enkephalin-like immunoreactive patches in the rostral than in the caudal striatum. Our data thus demonstrate that, by using computer-assisted morphometrical techniques, it is possible to describe a non-homogenous but overlapping distribution of tyrosine hydroxylase-like immunoreactive and dopamine- and cyclic AMP-regulated phosphoprotein-like immunoreactive patches in the rat neostriatum.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies to tyrosine hydroxylase and DARPP-32 were used to examine the spatial arrangement between mesostriatal dopamine projections and the reciprocal pathway from DARPP-32-containing neurons in the basal forebrain. Use of a double-labeling immunocytochemical procedure demonstrated that the mesostriatal and striatonigral pathways run in close proximity throughout the rostral mesencephalon and basal forebrain. The majority of descending axons immunoreactive for DARPP-32 appear to originate in the striatum, including the nucleus accumbens, and run through the internal capsule to innervate the globus pallidus, entopeduncular nucleus, and all subdivisions of the substantia nigra. The ventral tegmental area is sparsely invested with DARPP-32-immunoreactive axons. At all levels, there are also fascicles of DARPP-32-containing fibers which run ventromedial to the internal capsule in the medial forebrain bundle, and which are coextensive with ascending axons of the mesencephalic dopamine the internal capsule in the medial forebrain bundle, and which are coextensive with ascending axons of the mesencephalic dopamine cell groups. Tyrosine hydroxylase-immunoreactive axons are coextensive with DARPP-32-immunoreactive axons in the internal capsule entopeduncular nucleus, and globus pallidus, as well as much of the remainder of the basal forebrain. Although the main source of descending DARPP-32 immunoreactive axons would appear to be the striatum, other possible sources are also discussed.

Intrastriatal grafts of tissue obtained from the striatal or neocortical primordia of rat fetuses have been studied with respect to their intrinsic organization and connectivity using antibodies to DARPP-32 in combination with acetylcholinesterase (AChE) histochemistry, tyrosine hydroxylase (TH) immunocytochemistry, and anterograde and retrograde axonal tracing techniques. The striatal grafts were characterized by distinct patches of DARPP-32-immunoreactive neurons, which were identical to the densely AChE-positive patches stained in adjacent sections from the same specimens. The non-patch areas possessed only few DARPP-32-positive neurons and contained only sparse AChE-positive fibres. The cortical grafts, by contrast, contained no neurons with clear-cut DARPP-32-positivity and they exhibited a sparse, evenly distributed AChE fibre network, similar to that seen in the non-patch areas of the striatal grafts. The host dopaminergic afferents, as revealed by TH immunostaining, had grown selectively into the DARPP-32-positive patches in the striatal grafts, where they formed a dense terminal network around the DARPP-32-positive cell bodies. The non-patch areas, as well as the cortical grafts, received only sparse TH innervation. By contrast, the host cortical afferents, labelled by Phaseolus vulgaris leucoagglutinin from the host frontal cortex, were seen to extend into both the patch and non-patch areas of the striatal grafts. Transplant neurons projecting into the host brain were labelled by Fluoro-Gold injections into the ipsilateral host globus pallidus. These injections labelled large numbers of medium-sized neurons within the striatal grafts and the vast majority of them (over 85%) were confined to the DARPP-32-positive patches. Similar Fluoro-Gold injections labelled only few graft neurons in the cortical grafts. The results indicate that the striatal grafts are composed of a mixture of striatal and non-striatal tissue, and that the striatal graft compartment selectively establishes afferent and efferent connections with the host nigro-pallidal system. These graft connections demonstrate a remarkable specificity in the formation of graft - host connectivity. The results, moreover, suggest that developmental properties of the grafted striatal primordium are retained and expressed in the implanted cell suspension, and that the neuronal systems of the lesioned adult host brain, at least to some extent, remain responsive to growth regulating mechanisms normally operating during ontogenetic development.

Host afferents into fetal ventral mesencephalic tissue grafted to the neostriatum of adult rats have been studied by using anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and immunocytochemistry for serotonin (5-HT), Substance P (SP), and dopamine-adenosine 3':5'-monophosphate-regulated-phosphoprotein-32 (DARPP-32). Numerous fibers of cortical origin were detected in the transplants following multiple (11-15) iontophoretic injections of PHA-L into the frontal and anterior cingulate cortex. The labeled fibers occurred with an apparently random distribution throughout the graft tissue. Their overall density was lower than that of the surrounding striatum but similar to that found in the host nigra-ventral tegmental area. The majority of the PHA-L-labeled fibers in the grafts were thin and tortuous with varicosities or lateral clubs with terminal boutons. Dual labeling showed frequent close appositions between PHA-L-labeled terminals and dopamine-immunoreactive cell bodies. In parallel electron microscopy, synaptic contacts were observed between PHA-L-labeled terminals and unlabeled neuronal profiles in the graft. Other labeled fibers in the grafts were thick and smooth, corresponding probably to labeled myelinated axons observed in the electron microscope. These thick fibers were often seen to give off collaterals of the thin type. The virtual absence of such thick fibers in the normal striatal neuropil suggests that at least some of the cortical afferents to the grafts may have sprouted from axons normally projecting to diencephalic or brain stem regions. Serotonin fibers occurred in patches or as scattered single fibers in both deep and superficial portions of the nigral transplants. In the electron microscope some of these terminals were seen to establish synaptic contacts with nonimmunoreactive elements in the graft. These fibers were present also when the graft tissue had been pretreated with 5,7-dihydroxytryptamine at the time of transplantation. This treatment eliminated all 5-HT-containing neurons from the grafts without any noticeable adverse effect on the survival of the dopaminergic neurons. The serotonin fibers in the grafts were thus most likely of host origin. SP-positive fibers formed a dense plexus inside the grafts. Since many SP-positive cell bodies were visualized inside the transplant after colchicine pretreatment, it is unclear, however, whether any of these fibers were of host origin. Intrastriatal injections of PHA-L or DARPP-32 immunocytochemistry indicated that the deep portions of the nigral grafts were entirely devoid of host striatal afferents.(ABSTRACT TRUNCATED AT 400 WORDS)