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Microglia maintain normal brain function and support the brain’s response to disease and injury. The hippocampus is an area of focus for microglial study due to its central role in numerous behavioral and cognitive functions. Interestingly, microglia and related cells in the hippocampus and throughout the brain are distinct in male vs. female rodents, even in early life. Indeed, postnatal day (P)-dependent sex differences in number, density, and morphology of microglia-like cells have been reported in certain hippocampal subregions. For example, P3 female mice have more phagocytic microglia in dentate gyrus (DG) molecular layer (Mol) and CA1-3 stratum oriens (SO) regions vs. male mice, while P8 — but not P15 — male rats have more volume immunoreactive for markers of microglia-like cells (Iba1 and CD68) in the CA1 stratum radiatum (SR) vs. female rats. In the mouse, P10 is roughly equivalent to human term gestation, making it a common timepoint to study for many translationally-relevant neurobiological processes. However, sex differences in hippocampal microglia have not been examined in the P10 mouse hippocampus. In addition, key subregions of the hippocampus — CA3 SR, DG hilus — have not yet been assessed for sex differences in microglia. To address these knowledge gaps, we quantified Iba1+ cell densities and classified Iba1+ cell morphology in P10 male and female C57BL/6J mice. Four subregions in the bilateral anterior hippocampus were analyzed in 40-micrometer coronal sections: DG Mol (Mol), DG Hilus, CA1 SR and stratum lacunosum moleculare (CA1), and CA3 SR and stratum lucidum (CA3). Light microscope images (40x) were analyzed offline for Iba1+ cell density and morphology by an observer blind to sex. The morphology of each Iba1+ cell was used to place cells into one of four previously-published categories: Round or ameboid (round-ish soma, no processes), Stout (round-ish soma, short process), Thick (irregular soma with few, thick processes), or Thin (irregular soma with multiple thin processes). Analysis of Iba1+ cell density shows no difference between male and female mice in Mol, Hilus, CA3, or CA1 (male n=6, female n=7). However, morphology classification shows a sex-dependent difference in the Mol and Hilus, with female mice having a greater percentage of Thick Iba1+ cells vs. male mice (Mol, Hilus), and a lower percentage of Thin Iba1+ cells vs. male mice (Mol). With our analysis, it is unclear whether this greater percentage of thick and lower percentage of thin Iba1+ cells in the female vs. male hippocampus means Iba1+ microglia in female mice are ‘younger’ or ‘more active’ than those in male mice. However, these data are important as they reveal sex differences in Iba1+ microglia in the P10 mouse hippocampus. We discuss these results in the context of the large literature on sex differences in rodent microglia in the early postnatal period.

Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. Additionally, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches—Daun02 inactivation, FACS sorting of activated neurons and c-fos-GFP transgenic rats — that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools — c-fos-tTA mice and inactivation of CREB-overexpressing neurons — that have been used to study the role of neuronal ensembles in conditioned fear.

Cue-induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non-activated neurons during cue-induced heroin seeking after prolonged withdrawal. We trained rats to self-administer heroin for 10 days (6 h/day) and assessed cue-induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent-activated cell sorting (FACS) to purify Fos-positive and Fos-negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos-immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos-positive, but not Fos-negative, neurons. In support of these findings, double-label immunohistochemistry indicated substantial coexpression of neuropeptide Y (NPY)- and Arc-immunoreactivity in Fos-positive neurons. Our data indicate that cue-induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non-activated neurons.

Numerous studies with the neural activity marker Fos indicate that cocaine activates only a small proportion of sparsely distributed striatal neurons. Until now, efficient methods were not available to assess neuroadaptations induced specifically within these activated neurons. We used fluorescence-activated cell sorting (FACS) to purify striatal neurons activated during cocaine-induced locomotion in naive and cocaine-sensitizedcfos-lacZtransgenic rats. Activated neurons were labeled with an antibody against β-galactosidase, the protein product of the lacZ gene. Cocaine induced a unique gene expression profile selectively in the small proportion of activated neurons that was not observed in the nonactivated majority of neurons. These genes included altered levels of the immediate early genesarc,fosB, andnr4a3, as well as genes involved in p38 MAPK signaling and cell-type specificity. We propose that this FACS method can be used to study molecular neuroadaptations in specific neurons encoding the behavioral effects of abused drugs and other learned behaviors.

Learned associations between effects of abused drugs and the drug administration environment are important in drug addiction. Histochemical and electrophysiological studies suggest that these associations are encoded in sparsely distributed nucleus accumbens neurons that are selectively activated by drugs and drug-associated cues. Although correlations have been observed between nucleus accumbens neuronal activity and responsivity to drugs and drug cues, no technique exists for selectively manipulating these activated neurons and establishing their causal role in behavioral effects of drugs and drug cues. Here we describe a new approach, which we term the 'Daun02 inactivation method', that selectively inactivates a minority of neurons previously activated by cocaine in an environment repeatedly paired with cocaine to demonstrate a causal role for these activated neurons in context-specific cocaine-induced psychomotor sensitization in rats. This method provides a new tool for studying the causal roles of selectively activated neurons in behavioral effects of drugs and drug cues and in other learned behaviors.

Molecular analysis of brain tissue is greatly complicated by having many different classes of neurons and glia interspersed throughout the brain. Fluorescence-activated cell sorting (FACS) has been used to purify selected cell types from brain tissue. However, its use has been limited to brain tissue from embryos or transgenic mice with promoter-driven reporter genes. To overcome these limitations, we developed a FACS procedure for dissociating intact cell bodies from adult wild-type rat brains and sorting them using commercially available antibodies against intracellular and extracellular proteins. As an example, we isolated neurons using a NeuN antibody and confirmed their identity using microarray and real time PCR of mRNA from the sorted cells. Our FACS procedure allows rapid, high-throughput, quantitative assays of molecular alterations in identified cell types with widespread applications in neuroscience.