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. 2013 Apr 23:7:48.
doi: 10.3389/fncel.2013.00048. eCollection 2013.

Physiological temperature during brain slicing enhances the quality of acute slice preparations

Shiwei Huang  1 , Marylka Y Uusisaari
Affiliations

Physiological temperature during brain slicing enhances the quality of acute slice preparations

Shiwei Huang et al. Front Cell Neurosci. .

Abstract

We demonstrate that brain dissection and slicing using solutions warmed to near-physiological temperature (~ +34°C), greatly enhance slice quality without affecting intrinsic electrophysiological properties of the neurons. Improved slice quality is seen not only when using young (<1 month), but also mature (>2.5 month) mice. This allows easy in vitro patch-clamp experimentation using adult deep cerebellar nuclear slices, which until now have been considered very difficult. As proof of the concept, we compare intrinsic properties of cerebellar nuclear neurons in juvenile (<1 month) and adult (up to 7 months) mice, and confirm that no significant developmental changes occur after the fourth postnatal week. The enhanced quality of brain slices from old animals facilitates experimentation on age-related disorders as well as optogenetic studies requiring long transfection periods.

Keywords: acute slice preparation; cerebellar nuclei; cerebellum; mature animals.

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Figures

FIGURE 1
FIGURE 1
Comparison of the surface of acute coronal (top) and sagittal (bottom) mice (strain C57BL/6J 6w) cerebellar slices prepared with ice-cold (left) and warm (right) preparation methods. Patchable cells are indicated. All slices shown were prepared from mice aged 2.5 months, except for the sagittal slice cut at warm temperature (lower right) which was prepared from a 6-month old mouse. Note that in the warm-cut coronal slice, DCN cell bodies of both small (labeled “+”) and large (“*”) sizes are visible; in cold-cut coronal slices from old animals, most cells were small (labeled “+”). In cold-cut sagittal slices, hardly any healthy neurons in the Purkinje layer were found. In contrast, numerous patchable Purkinje neurons are easily seen in the warm-cut sagittal section. PN, Purkinje neuron layer; ML, molecular layer; GrC, granular cell layer.
FIGURE 2
FIGURE 2
Quantification of DCN slice healthiness with multi-electrode array recordings. Left: representative recordings with 60 planar extracellular electrodes from 7-month old mice from DCN slices prepared at cold (top, blue) and warm (bottom, red) temperatures. Each grid square represents 1 s of continuous recording (vertical axis: 100 pA); blank squares are electrodes with degraded recording quality. Electrodes in which spiking cells were detected are indicated with black, thick squares. A micrograph combined with a schematic drawing of a coronal cerebellar slice (right top panel) shows positioning of the multi-electrode array within the DCN. Right, middle and bottom panels: Comparisons of average and cumulative sums of the numbers of spiking cells detected per slice in < +30°C (+5 to 28°C, blue) and +30 to +34°C (red) conditions shows that when slices are prepared at warm temperatures, the probability of finding patchable neurons is increased. N = 23 (seven animals) slices cut in cold conditions, 10 (four animals) in warm.
FIGURE 3
FIGURE 3
(A) Intrinsic properties of juvenile (P18 – 28) Purkinje neurons are not affected by preparation temperature. Top panel: representative traces of voltage responses to step current injections in PNs from slices prepared in cold (blue) and warm (red) solutions. No differences beyond normal Purkinje neuron (PN) variability were apparent. Bottom panel: Left: Current-versus-firing frequency relation (blue and red dots) with linear fits (solid line) and 95% confidence interval (dashed lines) show similar gains of AP firing. Middle: population average APs (from all PNs from slices cut in cold (blue) and warm (red) solutions) show nearly indistinguishable shapes. Width of the trace represents the mean Vm ± SEM. Right: Comparison of intrinsic membrane properties (input resistance (Rm, MΩ), AP half-width (APHW, ms), AP amplitude (AP amp, mV), AP threshold (AP TH, mV), and AHP depth (mV) (right) show no statistically significant differences between cold (blue) and warm (red) preparation conditions (Rm: cold: 100.4 ± 6.5 MΩ, warm: 125.2 ± 18.0 MΩ; APHW: cold: 0.21 ± 0.01 ms; warm: 0.21 ± 0.01 ms; AP amp: cold: 77.9 ± 1.6 mV; warm: 76.8 ± 1.7 mV; APTH: cold: -48.6 ± 2.0 mV; warm: -44.9 ± 1.7 mV; AHP: cold: 15.0 ± 0.7 mV; warm: 16.6 ± 3.1 mV. P value for each data group >0.05. N: cold-cut n = 5, warm-cut n = 5. Rm, input resistance; AP, action potential; APHW, action potential half-width; ICMD, step command current; Cm, estimated neuronal capacitance (see methods). For averaged AP shapes, the thickness of the trace signifies ± SEM. Note that for bar graphs in A (bottom right), values for APHW and Ih-sag have been scaled (100× and 10×, respectively) for visual clarity. Also, note that in the left graph of the bottom panel, for correct comparison of current-to-firing relationships between neurons of possibly differing membrane resistances, the command current (ICMD) is scaled by multiplying by the measured Rm for each cell. (B). Morphology of a Purkinje neuron from a warm-cut slice (mouse age P18) filled with biocytin during electrophysiological recording. Compared to cold-cut, biocytin-filled Purkinje neurons (Jaeger, 2001; Bekkers and Häusser, 2007), there was no evidence suggesting that dissection temperature affects dendritic structure.
FIGURE 4
FIGURE 4
Warm preparation temperature allows patch-clamp recordings from mature and old DCN neurons. Top panel: Representative examples of voltage responses of DCN neurons to step current injections (two leftmost panels) as well as population average AP shapes in juvenile (less than P30) and mature (1–7 months old) animals are nearly indistinguishable. Bottom panel: Comparison of current-versus-firing (with linear polynomial fits and 95% prediction bounds), spike frequency accommodation (vertical bars denote frequency standard error values. All values are normalized to the first inter-spike interval firing frequency; a dashed line demarcates the starting value) and intrinsic membrane measures (Rm (MΩ), membrane time constant (Tau, ms), AP amplitude (AP amp, mV), AP threshold (AP TH, mV), AHP depth (AHP, mV), and Ih-sag (mV)) reveal no statistically significant differences between juvenile and old DCN neurons (Values: Rm: young: 101.5 ± 11.8 MΩ, old: 130.9 ± 17.9 MΩ; Tau, young: 13.6 ± 4.7 ms, old: 20.3 ± 2.7 ms; AP amp: young: 61.0 ± 3.4 mV; old: 63.6 ± 2.3mV; APTH: young: -48.6 ± 2.0 mV; old: -44.9 ± 1.7 mV; AHP: young: 24.0 ± 6.7 mV; old: 22.5 ± 1.2 mV; Ih-sag: young: 27.2 ± 8.9 mV, old: 28.9 ± 3.4 mV; P value for each data group >0.05). However, a slight decrease in mean AP half-width (APHW: young: 0.3 ± 0.01 ms; old: 0.26 ± 0.01 ms; P = 0.04) was, however, seen, possibly reflecting final maturation of the neuronal population. N = 5 GADnL neurons in young animals, 23 in old. Abbreviations: Rm, input resistance; AP, action potential; APHW, action potential half-width; ICMD, step command current; Cm, estimated neuronal capacitance (see methods). For averaged AP shapes, the thickness of the trace signifies ± SEM. Note that for correct comparison of current-to-firing relationships between neurons of possibly differing membrane resistances, the command current (ICMD) in bottom left panel is scaled by multiplying by with the measured Rm for each cell. Also note that in the bar graphs (bottom right), values for APHW and Ih-sag have been scaled (100× and 10×, respectively) for visual clarity.
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