Acute slice preparation for electrophysiology increases spine numbers equivalently in the male and female juvenile hippocampus: a DiI labeling study

Abstract

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. 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. 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. 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. We also show that the procedures used for in vitro electrophysiology also result in significant spine increases in the DG and CA1 subfields. These results demonstrate the utility of this refined DiI procedure for staining neuronal dendrites and spines. 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. 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.

A Student’s Guide to Neural Circuit Tracing

“…Interestingly, because they are not dependent on active transport, inorganic dyes may also be used for identification of projections in fixed tissue post mortem . Typically applied as crystals to the surface of formalin-fixed tissue blocks, highly lipophilic dyes such as DiI (red) and DiO (green) move evenly throughout cells in both anterograde and retrograde directions via the lipid portion of neuronal membranes, resulting in complete labeling of the soma and dendritic tree (Thanos et al, 1991, 1992; Köbbert et al, 2000; Boon et al, 2019; Trivino-Paredes et al, 2019). However, the lipophilic nature of carbocyanine dyes makes them difficult to use in conjunction with standard IHC protocols that use lipid-solubilizing detergents to facilitate antibody penetration (Elberger and Honig, 1990; Matsubayashi et al, 2008).…”

Primate neuronal connections are sparse in cortex as compared to mouse

“…Last, the majority of spine counting in primate neurons has been accompanied by prior electrophysiological characterization of neurons (Gilman et al, 2017;Luebke et al, 2004;Luebke and Rosene, 2003;Medalla and Luebke, 2015). However, the preparation of living brain slices can induce new spine formation, often twice the number relative to perfusion controls (Kirov et al, Cell Reports 36, 109709, September 14, 2021 11 Article ll OPEN ACCESS 1999, 2004Trivino-Paredes et al, 2019). These potential artifacts are particularly problematic in living brain slices chilled prior to recordings, a protocol used for most structural synaptic analyses of primates (Gilman et al, 2017;Luebke et al, 2004;Medalla and Luebke, 2015).…”

Quantification of Dendritic Spines Remodeling under Physiological Stimuli and in Pathological Conditions

“…To highlight the role of dendritic spines in various context-dependent conditions, we have summarized dendritic spine remodeling in physiological processes and upon stimulation ( Table 1 ) induced by postsynaptic receptor antagonism [ 15 ], postsynaptic receptor agonism [ 16 , 17 ], genetic modifications [ 18 , 19 , 20 ], Chemically induced LTP [ 21 , 22 , 23 , 24 , 25 ], LTD (long-term depression) [ 26 ], sensory experience [ 27 , 28 ], spatial memory and learning [ 29 ], physical environmental stimuli [ 30 , 31 , 32 ]. Pathological processes ( Table 2 ) were categorized into brain diseases underling neurodegenerative (Alzheimer’s disease [ 33 , 34 , 35 ], Parkinson disease [ 36 ], Fragile X Syndrome [ 37 , 38 ], Down Syndrome [ 39 ], Rett Syndrome [ 40 , 41 ], Autism Spectrum Disorder [ 42 ], Huntingtin Disease [ 43 ]), neuropsychiatric (Schizophrenia [ 44 ], Depression [ 45 , 46 , 47 ]), Stroke [ 48 ], Epilepsy [ 49 ], and infectious diseases such as Prion Disease [ 50 ], HIV infection [ 51 ], Influenza Infection [ 52 ], Toxoplasmosis [ 53 ] and Antiviral Responses [ 54 ].…”

“…Spines in acute brain slices after chemical fixation [ 97 ] are labeled with DiI [ 18 , 21 , 32 , 35 , 47 , 53 , 96 ] or Golgi staining [ 15 , 34 , 46 , 49 , 52 ]. The key element in dendritic spine staining is to apply an appropriate, gentle tissue fixation to assess the relevant effects on spine structure and eliminate cascades of biochemical processes affecting biophysical properties of neuronal membrane caused by cell death [ 32 , 96 , 98 , 99 ], therefore the vital condition of cells is an important factor in accurate spine analysis [ 97 , 100 ]. It is still not clear how in vitro, ex vivo, and in vivo models differ in speed of biochemical processes [ 97 , 100 , 101 , 102 ].…”

“…The experiments exhibit the nested hierarchical structure groups-animals-cells-dendrites-dendrite branches-spines. Thus, the nested statistical tests [ 16 ], or ANOVA test with post-hoc tests (such as Tuckey’s test [ 15 , 17 , 18 , 19 , 25 , 30 , 31 , 32 , 43 , 44 ]; unpaired t -test with Welch’s correction [ 18 , 21 , 24 , 47 ], two-tailed Student’s t -test [ 22 ]; Mann-Whitney test [ 20 , 23 , 28 , 35 , 37 ], Fisher’s least square difference [ 38 , 42 ] are used when applicable. Other statistical variants include Dunn’s Test [ 46 ], linear regression with lines fitted to the density plots for each cell [ 27 ], simple linear regression and multivariate linear regression [ 34 ]; Kolmogorov-Smirnov test combined with Student’s t test or Mann–Whitney U test [ 26 , 29 , 36 , 40 , 41 , 50 , 51 ], correlation analysis using Pearson’s correlation [ 34 , 49 ].…”