Isoxazole-9 reduces enhanced fear responses and retrieval in ethanol-dependent male rats

Abstract

Plasticity in the dentate gyrus (DG) is strongly influenced by ethanol, and ethanol experience alters long-term memory consolidation dependent on the DG. However, it is unclear if DG plasticity plays a role in dysregulation of long-term memory consolidation during abstinence from chronic ethanol experience. Outbred male Wistar rats experienced 7 weeks of chronic intermittent ethanol vapor exposure (CIE). Seventy-two hours after CIE cessation, CIE and age-matched ethanol-naïve Air controls experienced auditory trace fear conditioning (TFC). Rats were tested for cue-mediated retrieval in the fear context either twenty-four hours (24 hr), ten days (10 days), or twenty-one days (21 days) later. CIE rats showed enhanced freezing behavior during TFC acquisition compared to Air rats. Air rats showed significant fear retrieval, and this behavior did not differ at the three time points. In CIE rats, fear retrieval increased over time during abstinence, indicating an incubation in fear responses. Enhanced retrieval at 21 days was associated with reduced structural and functional plasticity of ventral granule cell neurons (GCNs) and reduced expression of synaptic proteins important for neuronal plasticity. Systemic treatment with the drug Isoxazole-9 (Isx-9; small molecule that stimulates DG plasticity) during the last week and a half of CIE blocked altered acquisition and retrieval of fear memories in CIE rats during abstinence. Concurrently, Isx-9 modulated the structural and functional plasticity of ventral GCNs and the expression of synaptic proteins in the ventral DG. These findings identify that abstinence-induced disruption of fear memory consolidation occurs via altered plasticity within the ventral DG, and that Isx-9 prevented these effects.

Keywords: CIE; CaMKII; Fos; Golgi-Cox; excitability; trace fear conditioning.

FIGURE 1

CIE enhances acquisition of TFC and retrieval of fear memories during protracted abstinence. (a) Schematic of experimental design and experimental groups used in the study, and timeline of blood collection for blood alcohol levels. Rats either experienced Air or CIE for 7 weeks. During the last week and a half of CIE, a subset of Air and CIE rats received vehicle or isoxazole-9 (Isx-9) injections (i.p.) for 12 days that extended into early withdrawal. A subset of vehicle and Isx-9 rats received one injection of BrdU to label actively dividing neural progenitor cells. Twenty-four hours after the BrdU injection, rats were trained on a TFC paradigm (b). (b,c) Schematic of TFC protocol (b) and retrieval (c). A subset of rats was tested for context retrieval and CS retrieval 24 hr, 10 days, or 21 days after TFC training (c). (d) Freezing, indicated as percent freezing responses, mean (±SE), during the 3-min baseline session. (e,f) Change in percent freezing, mean (±SE), from baseline during TFC acquisition during tone 1–5 (e) and trace 1–5 (f) sessions. (g,h) Percent freezing responses during context retrieval (g) and CS retrieval (h) from 24 hr, 10 days, and 21 days time points. Training and acquisition: n = 58 Air^v (−Isx-9), n = 58 CIE^v (−Isx-9), n = 40 Air^I (+Isx-9), and n = 39 CIE^I (+Isx-9). Retrieval 24 hr: n = 16 Air^v, n = 16 CIE^v, n = 12 Air^I, and n = 12 CIE^I. Retrieval 10 days: n = 16 Air^v, n = 16 CIE^v, n = 12 Air^I, and n = 12 CIE^I. Retrieval 21 days: n = 26 Air^v, n = 26 CIE^v, n = 16 Air^I, and n = 15 CIE^I. *p < 0.05 in d–f. Significance in (g,h, p < 0.05) is indicated by connected lines between groups

FIGURE 2

CIE alters structural plasticity of GCNs distinctly in dorsal and ventral regions of the DG. (a) Photomicrograph of Golgi-Cox-labeled cells in the dorsal DG with arrowhead pointing to a fully labeled GCN in the granule cell layer (GCL). Scale bar in (a) is 200 μm. (b,c) Higher magnification of Golgi-Cox-labeled cells in the GCL (b). One traced Golgi-Cox-labeled neuron with a Sholl ring (c) Each ring is 20 μm. (d–f) x–y graphical representation of 3D Sholl data, mean (±SE), of dorsal GCNs from 24 hr (d), 10 days (e), and 21 days (f) time points with number of intersections on y axis and distance from soma on x axis. (g–i) x–y graphical representation of 3D Sholl data of ventral GCNs from 24 hr (g), 10 days (h), and 21 days (i). (j) Example traces of GCNs from each group at the 21 days time point. *p < 0.05 CIE^v compared with Air^v group. Four GCNs were traced in the dorsal DG and four GCNs were traced in the ventral DG from each rat in each group. The data from four cells were averaged and used as one data for each rat. Number of rats- 24 hr: n = 4 Air^v (Air-Isx-9), n = 4 CIE^v (CIE-Isx-9), n = 4 Air^I (Air+Isx-9), and n = 4 CIE^I (CIE+Isx-9). 10 days: n = 4 Air^v, n = 4 CIE^v, n = 4 Air^I, n = 4 CIE^I. 21 days: n = 4 Air^v, n = 4 CIE^v, n = 4 Air^I, and n = 4 CIE^I

FIGURE 3

CIE distinctly alters the expression of proteins involved in glutamatergic signaling in the dorsal and ventral DG. (a–d) Schematic representation of the adult rat dorsal (a,b) and ventral (c,d) hippocampus modified from the rat brain atlas. Regions of tissue punches used for Western blotting are indicated as colored circles, with purple circles showing areas of the dorsal DG and blue circles showing areas of the ventral DG. (e) Sample images of proteins used for density analysis with molecular weights in kDa. Coomassie staining (Coom) was used as a loading control. (f–h) Density of protein expression, mean (±SE), for GluA1, pCaMKII, and tCaMKII in the dorsal DG from 24 hr (f), 10 days (g), and 21 days (h) time points. (i–k) Density of protein expression for GluA1, pCaMKII, and tCaMKII in the ventral DG from 24 hr (i), 10 days (j), and 21 days (k) time points. Number of rats- 24 hr: n = 10–12 Air^v (Air-Isx-9), n = 10–12 CIE^v (CIE-Isx-9), n = 10–12 Air^I (Air+Isx-9), and n = 10–12 CIE^I (CIE+Isx-9). 10 days: n = 10–12 Air^v, n = 10–12 CIEv, n = 10–12 AirI, and n = 10–12 CIEI. 21 days: n = 10–12 Air^v, n = 10–12 CIE^v, n = 10–12 Air^I, and n = 10–12 CIE^I

FIGURE 4

CIE reduces intrinsic excitability of ventral GCNs without altering excitability of dorsal GCNs. (a) Photomicrograph of the granule cell layer (GCL) in the dorsal dentate gyrus with a granule cell neuron patched for electrophysiology. Arrow points to a granule cell neuron. Hil, hilus. Image is captured at 400× magnification. (b) Representative sEPSC trace from a GCNs from dorsal (top) and ventral (bottom) DG from the Air group. (c,d) Group comparisons in GCNs demonstrated a main effect of DG subregion in sEPSC frequency (c) and amplitude (d). No significant differences were noted between experimental groups within each DG subregion in sEPSC amplitude or frequency. (e) Representative traces of action potentials elicited by depolarizing current injection (120 pA) from dorsal GCNs from Air (left panel) and CIE (right panel) groups. (f,g) Graphical relationship between the number of spikes elicited by increasing current injections in current-clamp recordings from GCNs from all groups. (h) Representative traces of action potentials elicited by depolarizing current injection (120 pA) from ventral GCNs from Air (left panel) and CIE (right panel) groups. (I,j) Graphical relationship between the number of spikes elicited by increasing current injections in current-clamp recording from GCNs from all groups. *p < 0.05 versus CIE condition. Data are expressed as mean (±SE). GCNs (number of rats/number of cells): 21 days dorsal: n = 7/11 Air^v, n = 7/13 CIE^v, n = 4/13 Air^I, and n = 3/4 CIE^I. 21 days ventral: n = 7/7 Air^v, n = 7/6 CIE^v, n = 4/11 Air^I, and n = 3/15 CIE^I

FIGURE 5

TFC and Isx-9 enhance neuronal activity in the ventral GCNs and this effect is dampened in CIE rats. (a) Schematic representation of a sagittal section through the rat brain hippocampus at bregma −6.3. The area in the box represents the area in the photomicrograph in b. (b,c) Representative photomicrographs of Fos (b,d) and BrdU (c)-labeled cells in the granule cell layer (GCL) of the dentate gyrus. Arrowheads point to immunoreactive cells. Scale bar in d is 20 μm, in c 15 μm and in b is 200 μm. (e,f) Quantitative data, mean (±SE), of BrdU (e) and Fos (f) cells in each experimental group. #, main effect and *, significance by post hoc analysis. Number of rats: behavior naïve- n = 3–7 Air, n = 4 CIE; TFC-Isx-9- n = 12 Air, n = 11–12 CIE; TFC+Isx-9- n = 8 Air, n = 6–8 CIE