Neural bases for addictive properties of benzodiazepines

Abstract

Benzodiazepines are widely used in clinics and for recreational purposes, but will lead to addiction in vulnerable individuals. Addictive drugs increase the levels of dopamine and also trigger long-lasting synaptic adaptations in the mesolimbic reward system that ultimately may induce the pathological behaviour. The neural basis for the addictive nature of benzodiazepines, however, remains elusive. Here we show that benzodiazepines increase firing of dopamine neurons of the ventral tegmental area through the positive modulation of GABA(A) (gamma-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on alpha1-containing GABA(A) receptors expressed in these cells, triggers drug-evoked synaptic plasticity in excitatory afferents onto dopamine neurons and underlies drug reinforcement. Taken together, our data provide evidence that benzodiazepines share defining pharmacological features of addictive drugs through cell-type-specific expression of alpha1-containing GABA(A) receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing alpha1 may be devoid of addiction liability.

Figure 1. BDZ-evoked synaptic plasticity is abolished in α1(H101R) mutant mice

a, Top panel; normalized AMPAR-EPSCs obtained at -65, 0 and +35 mV in slices from WT mice i.p. injected with saline, MDZ (0.5 mg/kg) or Mor (15 mg/kg) 24 h prior to sacrifice. Middle panel; corresponding iv-curves. Bottom panel; bar graphs represent group data for the RI. F_(2;21) = 9.08. b, AMPAR-EPSCs, iv-curves and RI (top, middle and bottom panel, respectively) observed when ACSF or MDZ were injected into the VTA in WT mice. t₍₁₁₎ = 5.43. c, Similar experiments performed with α1(H101R) mice. Note that Mor induces a rectification that is similar in WT and mutant mice. F_(2;16) = 17.88. d, Similar experiments performed with α1(H101R) mice when MDZ was injected intra-VTA. n = 6-10.

Figure 2. Synaptic plasticity evoked by α1-subunit selective compounds

a, Normalized AMPAR-EPSCs obtained at -65, 0 and +35 mV in slices from WT mice injected with ZOL (5 mg/kg i.p.), L-838 417 (10 mg/kg i.p.) and MDZ together with Flu (5 mg/kg), 24 h prior to sacrifice. b, Corresponding iv-curves. c, Bar graphs representing group data for the RI. F_(2,19) = 28.97. n = 6-8.

Figure 3. α1 is selectively expressed in GABA neurons of the VTA

a, Immunohistochemical staining for tyrosine hydroxylase (TH, red) and ༟1 (blue) in VTA slices of GAD67-GFP (green) knock-in mice. Concentric pie charts represent the fraction of α1-positive cells (inner segment), and quantification of the two cell types (outer segment, n = 4 mice). Overlap between inner and outer segments represents colocalization. b, Example trace of mIPSCs recordings in GABA and DA neurons obtained in slices from WT mice. c, Representative averaged mIPSC trace from a GABA and a DA neuron. The overlay shows the difference in kinetics when the two currents are normalized to the average mIPSC peak amplitude. d, Box-plots represent group data for charge transfer and amplitude of mIPSCs obtained from GABA and DA neurons in slices from WT mice. t₍₇₅₎ = 7.55 and t₍₇₅₎ = 3.16, respectively. (n = 25-48). e, Representative average traces of mIPSCs before (solid line) and after (dotted line) application of MDZ (100 nM) in slices from WT and α1(H101R) mice. f, Corresponding box-plots representing group data for relative increase in charge transfer and frequency after MDZ bath-application. t₍₁₄₎ = 3.06 and t₍₁₄₎ = 3.23. n = 6-10.

Figure 4. The total current generated by sIPSC in DA neurons is decreased by MDZ

a, Example trace of sIPSCs recordings in GABA and DA neurons obtained before and after application of MDZ in slices from WT and α1(H101R) mice. sIPSCs were abolished with picrotoxin (PTX, 100 μM, not shown). b, Group data for the relative increase in the overall charge transfer (1 min) after MDZ bath-application. Note that in WT mice the total current in DA neurons decreases with MDZ application while in α1(H101R) mice there is an increase. GABA/WT vs GABA/α1(H101R) t₍₉₎ = 6.39, DA/WT vs DA/α1(H101R) t₍₁₅₎ = 5.50. n = 6-7.

Figure 5. Opposing effects of MDZ on in vivo firing rates of DA and GABA neurons

a, Representative extracellular single unit recording of a DA neuron during the i.v. injection of MDZ (0.5 mg/kg) in WT mice. Corresponding firing frequency plot (lower panel; Flu 1 mg/kg). b, Same experiment in α1(H101R) mice. c, Same experiment as in a) while monitoring a GABA neuron. d, Response of a GABA neuron to MDZ in an α1(H101R) mouse. White bars indicate time windows of traces shown above. e, Normalized firing rate of DA neurons in response to MDZ as a function of the basal activity in WT and α1(H101R) mice. WT/ α1(H101R) : F_(2;23) = 10.63. f, Corresponding plot with the results obtained in GABA neurons. Notice that 3 out of 5 neurons were completely silenced, which precluded fitting. g, Box-plots representing group data for relative change in firing rate. WT DA/ α1(H101R) DA: t₍₂₃₎ = 2.70, WT GABA/ α1(H101R) GABA t₍₁₂₎ = 4.60. h, Normalized firing rate in response to i.v. injection of Mor (5 mg/kg) as a function of the basal activity in WT and α1(H101R) mice. Solid lines: regression curves; shaded area: 95% confidence intervals. n = 5-15.

Figure 6. Oral self-administration of MDZ

a, Protocol for behavioral experiment. b, Total consumption successively with water, sucrose, and MDZ (0.005 mg/ml) + sucrose (4 %) in WT mice (black) and α1(H101R) mice (red). Note that WT and α1(H101R) mice drink similar amounts of liquids. c, Relative MDZ consumption in WT and α1(H101R) mice. d, Corresponding box plots for relative average consumption of MDZ at days indicated. n = 12-18 mice in 4-6 cages. F_(3;16) = 5.39