Repeated social defeat stress-induced sensitization to the locomotor activating effects of d-amphetamine: role of individual differences.

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.

Cocaine-induced proliferation of dendritic spines in nucleus accumbens is dependent on the activity of cyclin-dependent kinase-5.

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.

Altered dendritic spine density in animal models of depression and in response to antidepressant treatment.

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.

Impaired conditioned taste aversion learning in spinophilin knockout mice.

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.

Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5.

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.

Chronic fluoxetine administration to juvenile rats prevents age-associated dendritic spine proliferation in hippocampus.

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 regulates the formation and function of dendritic spines.

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.

Quantitative immunocytochemistry of DARPP-32-expressing neurons in the rat caudatoputamen.

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.

DARPP-32: regulator of the efficacy of dopaminergic neurotransmission.

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.

Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines.

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.

Co-localization of the D1 dopamine receptor in a subset of DARPP-32-containing neurons in rat caudate-putamen.

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.

Differential expression of protein phosphatase 1 isoforms in mammalian brain.

Rat cDNAs encoding neuronal isoforms of protein phosphatase 1 (PP1) were isolated and their primary structures elucidated. The derived amino acid sequences allowed us to design synthetic C-terminal peptides that were used to raise antibodies. Isoform-specific anti-peptide antibodies against PP1 alpha and PP1 gamma 1 were used to investigate the tissue distribution of PP1 isoforms by immunoblotting. Both isoforms were ubiquitously expressed in mammalian tissues, with the highest levels being observed in brain. Of all neuronal tissues examined, PP1 alpha and PP1 gamma 1 were found to be most abundantly expressed in the striatum. Lesion experiments with kainic acid indicated that both the alpha and the gamma 1 isoforms of protein phosphatase 1 were relatively enriched in the medium-size spiny neurons of the striatum. "In situ" hybridization to rat brain slices using highly sensitive riboprobes also showed PP1 alpha, PP1 beta, and PP1 gamma 1 to be widely expressed in mammalian brain. However, some interesting differences were observed. For example, PP1 alpha and PP1 gamma 1 were found to be expressed in the striatum, where DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000 Da) is also known to be highly expressed. PP1 beta appeared to be relatively less abundant in the same cells, as judged both by "in situ" hybridization and by the apparent absence of PP1 beta clones from the striatal cDNA libraries used.

The alpha and gamma 1 isoforms of protein phosphatase 1 are highly and specifically concentrated in dendritic spines.

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.

Immunocytochemical localization of amyloid precursor protein in rat brain.

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.

Immunocytochemical localization of DARPP-32, a dopamine and cyclic-AMP-regulated phosphoprotein, in the primate brain.

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, a dopamine and cyclic AMP-regulated phosphoprotein, is present in corticothalamic neurons of the rat cingulate cortex.

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.

Localization of the MARCKS (87 kDa) protein, a major specific substrate for protein kinase C, in rat brain.

The localization of MARCKS (myristoylated, alanine-rich C-kinase substrate), a major specific substrate for protein kinase C, has been studied in the rat brain. Light microscopic immunocytochemistry and biochemical analysis demonstrated that the protein is widespread throughout the brain and enriched in certain regions, including the piriform and entorhinal cortices, portions of the amygdaloid complex, the intralaminar thalamic nuclei, the hypothalamus, the nucleus of the solitary tract, nucleus ambiguus, and many catecholaminergic and serotonergic nuclei. Electron microscopic analysis revealed immunoreactivity in axons, axon terminals, small dendritic branches, and occasionally in dendritic spines. In neuronal processes, immunoreactivity was particularly prominent in association with microtubules, but reaction product was also seen in cytosol and adjacent to plasma membranes. No reaction product was observed in large dendrites, somata, or nuclei. A population of strongly immunoreactive glial cells was also observed. Many of these glial cells were morphologically similar to microglial cells, although some resembled astrocytes. In glial cells, both cytoplasm and plasma membranes were heavily labeled. The distribution of the MARCKS protein did not coincide precisely with the distribution of any of the subspecies of protein kinase C. The results indicate that the MARCKS protein is expressed in the majority of cell types in the CNS, and they suggest that the protein may be involved both in glial cell functions and in neuronal functions involving cytoskeletal elements in small dendritic branches and axon terminals.

Distribution of DARPP-32 in the basal ganglia: an electron microscopic study.

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.