The mechanisms involved in spine generation are important to understand, because a number of sex-specific differences in synaptic plasticity have been reported in a variety of neuropathological processes that range from fetal alcohol spectrum disorders (FASD) to Fragile-X syndrome ( Bostrom et al. For instance, gonadotropin-releasing hormone (GnRH) can regulate estradiol synthesis, spine density, and the expression of synaptic proteins through both genomic and nongenomic mechanisms ( Fester et al. That is, ligand binding to a nuclear receptor can produce genomic effects by altering the transcription rate of target genes however, these same receptors can also have nongenomic effects, such as propagating signal transduction through a kinase pathway or activating G proteins ( Wilkenfeld et al. This is an important consideration, because sex hormones act throughout the entire brain of males and females, and they can exert their effects through both genomic and nongenomic receptor actions ( McEwen and Milner 2017). Thus hormonal variation could lead to sex-specific differences in spine dynamics. The shape and number of synapses can also be modulated by hormones, and, in particular, dendritic spines in the brain have been shown to vary in density across the estrous cycle ( Gould et al. Thus spine shape and numbers have become critical metrics for synaptic physiology related to learning and memory processes ( Lisman et al. In turn, the morphology of individual spines has been shown to change dramatically in response to both activity-dependent and -independent mechanisms ( Harris and Stevens 1988, 1989 Kasai et al. 2016 Rochefort and Konnerth 2012 Uteshev et al. Interestingly, the morphology of dendritic spines is highly variable, and it is thought that their structure (i.e., spine head width, spine neck length and width) influences their neurophysiological properties ( Rangamani et al. Dendritic spines are small (1–2 μm) membrane protrusions that are present on most neurons in the brain and are believed to be a major component of learning and memory processes ( Bailey et al. The ability of the brain to store and process information is thought to depend on the plastic nature of synapses, which are composed of a presynaptic axon terminals, dendritic postsynaptic spines, and associated glia ( Araque et al. NEW & NOTEWORTHY This study introduces a new DiI technique that elucidates differences in spine numbers in juvenile female and male hippocampus, and shows that slice preparations for hippocampal electrophysiology in vitro may mask these differences. They also show, for the first time, that in vitro electrophysiology slice preparations enhance spine numbers on hippocampal cells equivalently in both juvenile females and males. These results demonstrate the utility of this refined DiI procedure for staining neuronal dendrites and spines. We also show that the procedures used for in vitro electrophysiology also result in significant spine increases in the DG and CA1 subfields. We show that the preparation of sections for electrophysiology produces significant increases in spines in sections obtained from females, similar to that observed in males. In the current study we use microcrystals of the lipophilic carbocyanine dye DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) to stain individual neurons in the CA1 and dentate gyrus (DG) hippocampal subfields of postnatal day 21 male and female rats. To date, several studies have shown that the process of making slices from male animals can induce synaptogenesis in cornu ammonis area 1 (CA1) pyramidal cells, but there is a paucity of data for females and other brain regions. Hippocampal slices are widely used for in vitro electrophysiological experiments to study underlying mechanisms for synaptic transmission and plasticity, and there is a growing appreciation for sex differences in synaptic plasticity.