Agony of the very unlikely "addicts"

http://www.dailymail.co.uk/health/article-1259892/Thousands-60s-hooked-tranquillisers-turned-virtual-zombies.html

This is so sad!  I think that is what has happened with a lot of us.  The doctors are often too quick to prescribe for a problem that may be better treated by something else.

 

The other thing that struck me is that so many people over 60 are stuck on benzo's.  My psych doc that started me on K said not to worry because lots of people take this stuff for 10, 20 or more years.  Of course he didn't tell me anything about the quality of thier lives after having been on for that long.

 

So sad, at least in Britain they are making efforts to curb benzo addiction.  Thanks for sharing!

Yes, thanks for this article. Just terrible.

 

England is so much further ahead of us, but then I read Barry Haslam is stepping down in the UK as he thinks the government is pushing involuntary addiction into the addiction arena. They still don't get it.

 

How do you get officials to understand what is being called "therapeutic doses"  are what's addicting patients? Doctors calling .5 mg of klonopin daily for a year as a "therapeutic dose" is misleading the officials. IMO.

Here is the actual study and its results for anyone with the time and energy for a brain squeeze...

 

Article

 

Nature 463, 769-774 (11 February 2010) | doi:10.1038/nature08758; Received 9 July 2009; Accepted 2 December 2009

 

Neural bases for addictive properties of benzodiazepines

 

Kelly R. Tan1, Matthew Brown1,6, Gwenaël Labouèbe1,6, Cédric Yvon1,6, Cyril Creton1, Jean-Marc Fritschy2, Uwe Rudolph3 & Christian Lüscher1,4,5

 

  1. Department of Basic Neurosciences, Medical Faculty, University of Geneva, CH-1211 Geneva, Switzerland

  2. Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland

  3. Laboratory of Genetic Neuropharmacology, McLean Hospital and Department of Psychiatry, Harvard Medical School, Belmont, Massachusetts 02478, USA

  4. Clinic of Neurology, Department of Clinical Neurosciences, Geneva University Hospital, CH-1211 Geneva, Switzerland

  5. Geneva Neuroscience Center, CH-1211 Geneva, Switzerland

  6. These authors contributed equally to this work.

 

Correspondence to: Christian Lüscher1,4,5 Correspondence and requests for materials should be addressed to C.L. (Email: christian.luscher@unige.ch).

 

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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 GABAA (γ-aminobutyric acid type A) receptors in nearby interneurons. Such disinhibition, which relies on α1-containing GABAA 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 α1-containing GABAA receptors in the ventral tegmental area. The data also indicate that subunit-selective benzodiazepines sparing α1 may be devoid of addiction liability.

 

Addictive drugs can be classified into three groups, according to the cellular mechanism through which they increase mesolimbic dopamine (DA)1. Opioids, cannabinoids and the club drug γ-hydroxybutyrate reduce release from inhibitory afferents onto DA neurons, through their respective G-protein-coupled receptors on GABA neurons. These substances activate pre- and postsynaptic receptors, indirectly increasing the firing rate of DA neurons, a mechanism defined as disinhibition. Nicotine, as a member of the second group, directly depolarizes DA neurons by activating α4β2-containing acetylcholine receptors, whereas the third group targets DA transporters (for example, cocaine and amphetamines). It remains unclear whether these mechanisms can account for the addiction liability of benzodiazepines (BDZs)2, which are positive modulators of GABAA receptor (GABAAR) function.

 

As well as increasing mesolimbic DA, another common feature of all addictive drugs studied so far is that they trigger adaptive synaptic plasticity in the ventral tregmental area (VTA)3. Hours after the initial exposure, excitatory afferents onto DA neurons of the VTA are strengthened, in part by the insertion of GluR2-lacking AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors)4, 5, 6. To test whether a similar mechanism is elicited by BDZs, we examined whether a single injection of BDZ would, as well as causing an increase in the AMPA/NMDA ratio7, also induce a change in the slope of the current–voltage (I–V)-curve of evoked excitatory postsynaptic currents (EPSCs). Such rectification reflects the presence of GluR2-lacking AMPARs, which are calcium permeable and blocked by polyamines at positive potentials.

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BDZ-evoked plasticity in dopamine neurons

 

In slices obtained 24 h after the intraperitoneal (i.p.) injection of midazolam (MDZ), diazepam or flunitrazepam, the rectification index (RI = EPSC-65 mV/EPSC+35 mV) was significantly higher than in slices from saline-injected controls (Fig. 1a and Supplementary Fig. 2). Similar rectification was measured after an injection of morphine, a member of the class of drugs that cause disinhibition of DA neurons8. The BDZ antagonist flumazenil blocked rectification when co-injected with MDZ, but was without effect when co-injected with a control saline solution (Fig. 2 and Supplementary Fig. 2). The adaptive plasticity induced by systemic BDZs was also observed 24 h after local application of MDZ into the VTA by stereotactic injection (0.5 μl of an 8 mg ml-1 solution over 10 min; Fig. 1b). Thus, BDZ-dependent effects on VTA circuitry are sufficient to induce this cellular hallmark of addictive drugs.

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

Figure 1 : BDZ-evoked synaptic plasticity is abolished in |[agr]|1(H101R) mutant mice. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

 

a, Top, normalized AMPAR-EPSCs obtained at -65, 0 and +35 mV in slices from wild-type (WT) mice after i.p. injection with saline, MDZ (0.5 mg kg-1) or morphine (MOR; 15 mg kg-1), 24 h before being euthanized. Middle, corresponding I–V curves. Bottom, bar graphs represent group data for the rectification index (RI). F(2;21) = 9.08. b, AMPAR-EPSCs, I–V curves and rectification index (top, middle and bottom panels, respectively) observed when ACSF or MDZ was injected into the VTA in wild-type mice. t(11) = 5.43. c, Similar experiments performed with α1(H101R) mice. Note that morphine induces a rectification that is similar in wild-type 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. Data are mean ± s.e.m.; **P < 0.01, ***P < 0.001.

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

Figure 2 : Synaptic plasticity evoked by |[agr]|1-subunit-selective compounds. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

 

a, Normalized AMPAR-EPSCs obtained at -65, 0 and +35 mV in slices from wild-type mice injected i.p. with ZOL (5 mg kg-1), L-838 417 (10 mg kg-1) and MDZ together with flumazenil (FLU; 5 mg kg-1), 24 h before being euthanized. b, Corresponding I–V curves. c, Bar graphs representing group data for the rectification index. F(2,19) = 28.97; n = 6–8. Data are mean ± s.e.m.; ***P < 0.001.

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BDZs bind to GABAARs at the interface between α and γ subunits9 in a subunit-dependent manner. GABA neurons in many parts of the brain express the α1 subunit isoform10, whereas midbrain DA neurons lack α1 but express α2, α3 and α4 subunit isoforms11. Thus, the addictive potential of BDZs might rely on the potentiation of α1-containing GABAARs, which would selectively inhibit GABA neurons and lead to disinhibition of DA neurons. To test this idea, we examined whether MDZ (that is, a rapidly acting, non-selective BDZ with a very strong brain uptake12) has an effect in mice with a point mutation (H101R) in the α1 subunit that disrupts the site where BDZs normally bind13. In α1(H101R) mice, an i.p. MDZ injection no longer had an effect on the rectification index of AMPAR EPSCs in DA neurons (Fig. 1c). This was not due to a general loss of adaptive plasticity, as morphine still caused a strong rectification. Moreover, stereotactic injections of MDZ into the VTA also failed to elicit rectifying AMPAR-mediated EPSCs in α1(H101R) mice, whereas control injections of artificial cerebrospinal fluid (ACSF) were without effect in either genotype (Fig. 1d). Furthermore, i.p. injection of MDZ increased the AMPA/NMDA ratio in wild-type but not in α1(H101R) mice (Supplementary Fig. 3).

 

We next used pharmacological tools to confirm the involvement of α1. Zolpidem (ZOL) is a non-classical BDZ selective for α1-containing GABAARs14, whereas the experimental compound L-838 417 does not modulate receptors that contain α1 (ref. 15). We therefore tested whether ZOL and L-838 417 could evoke synaptic plasticity in DA neurons. We found that a single injection of ZOL led to rectifying AMPAR-mediated EPSCs, whereas L-838 417 did not affect the I–V curve (Fig. 2). Taken together with the results in α1(H101R) mice described earlier, we conclude that BDZ-evoked synaptic plasticity depends on α1-containing GABAARs within the VTA.

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Cell type-specific expression of α1

 

To identify α1-expressing cells in the VTA, we next carried out immunohistochemical staining for tyrosine hydroxylase and the α1 subunit isoform in GAD67 green fluorescent protein (GFP) mice (Fig. 3a). These experiments confirmed that α1 was expressed mainly in GFP-positive neurons, but not in tyrosine-hydroxylase-positive DA neurons. Quantification showed that 81% of the GABA neurons contained the α1 subunit isoform, although this was only the case in 7% of the DA neurons (Fig. 3a, inset). We also observed α1-staining that could neither be associated to tyrosine-hydroxylase-positive nor GAD67–GFP-expressing cells. This may reflect the pool of the so-called tertiary cells that are neither DA- nor GABA-neurons16, 17, or could be due to detectability limits in fine processes.

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

Figure 3 : |[agr]|1 is selectively expressed in GABA neurons of the VTA. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

 

a, Immunohistochemical staining for tyrosine hydroxylase (TH, red) and α1 (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 mIPSC recordings in GABA and DA neurons obtained in slices from wild-type 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 wild-type mice. The box represents the median and interquartile range, the top and bottom vertical bars denote the 90th and 10th percentile. 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 wild-type 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. Data are mean ± s.e.m.; **P < 0.01, ***P < 0.001.

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To assess the functional consequences of this cell-type-specific isoform expression for inhibitory transmission, we characterized miniature inhibitory postsynaptic currents (mIPSCs) in the presence of the glutamate receptor blocker kynurenic acid to isolate GABAAR-mediated currents (Fig. 3b, c). On average, mIPSCs in GABA neurons were slower and bigger than those in DA neurons, leading to a significantly larger charge transfer in the former (Fig. 3d). This difference was of similar magnitude in wild-type and α1(H101R) mice (Supplementary Fig. 4), in line with previous reports13 that baseline transmission in mutant mice is normal. Moreover, the frequency of mIPSCs, as well as the multiplicity factor (Supplementary Fig. 4c and see Methods for detailed description), were similar in GABA and DA neurons in both genotypes. Although this approach has its limitations18, it suggests that the number of inhibitory synapses is in the same range in the two cell types. To confirm further that synapses on GABA neurons express α1-containing GABAARs, we tested for effects of MDZ on charge transfer and frequency of mIPSCs in wild-type and α1(H101R) mice. In DA neurons, MDZ significantly increased the charge transfer and decreased the mIPSC frequency in both genotypes. In GABA neurons, MDZ increased the charge transfer and decreased mIPSC frequency in slices from wild-type mice, but was without effect on mIPSCs recorded in slices from α1(H101R) mice (Fig. 3e, f and Supplementary Fig. 5a). As expected, MDZ had no effect on mIPSC amplitude in either cell type or genotype19 (Supplementary Fig. 5b). The observation that the mIPSC frequency is reduced by BDZs except in GABA neurons of α1(H101R) mice is surprising at first, but could reflect presynaptic GABAARs. In fact, such receptors have been described in the VTA, which after activation reduce the release probability20.

 

Because DA neurons express a set of many subunit isoforms11, the identification of the molecular composition of the GABAARs is difficult. Most DA neurons actually express the α3 subunit isoform (96%, Supplementary Fig. 6). Notably, most GABA neurons do not express the α3 subunit isoform (70%) even though significant heterogeneity was observed. In heterologous expressed systems, currents of α1-containing receptors are smaller than those of α3-containing receptors21. This, however, does not apply to DA neurons in the VTA, because in α3 knockout mice currents are reduced by only 50%22. Our results establish that in α1(H101R) mice, endogenous GABAA-mediated synaptic transmission is normal, whereas the positive modulation of MDZ is abolished in GABA neurons, because the α1 subunit isoform is selectively expressed in these cells.

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Cellular determinants of disinhibition

 

In wild-type mice, mIPSCs in both GABA and DA neurons were enhanced by BDZs. However, when BDZs are administered while transmission is intact, the extent of current amplification in DA neurons depends on the frequency of synaptic events, which originate in the interneurons upstream. We therefore monitored the effect of MDZ on spike-driven, spontaneous IPSCs (sIPSCs) in DA neurons (Fig. 4). Although, the charge transfer of sIPSCs on average increased after MDZ (in line with the mIPSC data), there was a strong reduction of the frequency of spike-driven events in DA neurons (Supplementary Fig. 7). As a result, when we integrated the charge transfer of sIPSCs over time before and after the application of MDZ (relative total current), we found a significant decrease (Fig. 4b). Because interneurons are efficiently inhibited by MDZ, fewer spikes are generated, strongly decreasing the number of sIPSCs, an effect that predominates over the MDZ amplification of the individual event. In α1(H101R) mice, in contrast, we observed an increased total current in DA neurons because the GABA neurons were insensitive to MDZ. In summary, in wild-type mice, MDZ led to a net decrease of the total inhibitory current in DA neurons, which could be sufficient to cause their disinhibition (see Supplementary Fig. 1 for schematics).

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

Figure 4 : The total current generated by sIPSCs in DA neurons is decreased by MDZ. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

 

a, Example trace of sIPSC recordings in GABA and DA neurons obtained before and after application of MDZ in slices from wild-type and α1(H101R) mice. sIPSCs were abolished with picrotoxin (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 wild-type mice the total current in DA neurons decreases with MDZ application, whereas in α1(H101R) mice there is an increase. GABA/WT versus GABA/α1(H101R) t(9) = 6.39, DA/WT versus DA/α1(H101R) t(15) = 5.50; n = 6–7. Box plot designations are as in Fig. 3d; ***P < 0.001.

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We therefore tested the effect of MDZ on the firing rate of DA neurons in the VTA by performing extracellular single-unit recordings in vivo. When the drug was injected into the tail vein of wild-type mice, we recorded a significant increase in the firing rate that was reversed by flumazenil (Fig. 5a, e, g). In stark contrast, no such disinhibition could be observed in α1(H101R) mice (Fig. 5b, e, g). In line with a disinhibition model, the data in the DA neurons were mirrored by the observations in GABA neurons. MDZ caused an inhibition of the spontaneous firing rates, at times leading to complete spike suppression (Fig. 5c, f, g). In α1(H101R) mice, MDZ did not significantly affect firing in GABA neurons (Fig. 5d, f, g). The specificity of these findings are further demonstrated by the observation that in mice in which a different α subunit isoform had been mutated (α3(H126R) mice)23, MDZ caused an increase in the firing rate of DA neurons comparable to wild-type mice (Supplementary Fig. . The magnitude of this increase was inversely related to the basal firing rate, which further indicates a disinhibition model (Fig. 5e). Moreover, in α1(H101R) mice, disinhibition of DA neurons was observed with morphine, an effect that was also inversely correlated to the basal firing rate (Fig. 5h). Although anaesthesia may modify the overall distribution of firing rates and therefore the magnitude of the disinhibition, the mean basal firing rates observed here were comparable to values recorded in freely moving animals24, 25.

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

Figure 5 : Opposing effects of MDZ on in vivo firing rates of DA and GABA neurons. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

 

a, Representative extracellular single-unit recording of a DA neuron during the intravenous (i.v.) injection of MDZ (0.5 mg kg-1) in wild-type mice. Corresponding firing frequency plot (bottom panel; flumazenil 1 mg kg-1). 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 wild-type and α1(H101R) mice. Wild-type/α1(H101R) F(2;23) = 10.63. f, Corresponding plot with the results obtained in GABA neurons. Note that three out of five neurons were completely silenced, which precluded fitting. g, Box plots representing group data for relative change in firing rate. Wild-type DA/α1(H101R) DA t(23) = 2.70, wild-type GABA/α1(H101R) GABA t(12) = 4.60. Box plot designations are as in Fig. 3d; *P < 0.05, ***P < 0.001. h, Normalized firing rate in response to i.v. injection of morphine (5 mg kg-1) as a function of the basal activity in wild-type and α1(H101R) mice. Solid lines: regression curves; shaded area: 95% confidence intervals; n = 5–15.

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Self-administration of midazolam

 

The results demonstrate that α1-containing GABAARs mediate the increase of mesolimbic DA in response to BDZs. Furthermore, DA antagonists can reduce self-administration of and preference to these drugs26, 27. We therefore tested the effect of the α1 subunit isoform on oral self-administration of MDZ, by offering the mice a free choice of two drinking solutions (Fig. 6a). During the first 3 days the two bottles contained water. Sucrose was then added to both bottles to mask any bitter tastes. This led to an increase in the overall consumption, but no particular preference. Finally, MDZ was added to one of the two bottles. During the test period with MDZ, the total consumption did not change in either genotype (Fig. 6b). A preference for the MDZ solution developed rapidly in wild-type mice, but not in α1(H101R) mice (Fig. 6c, d). Wild-type mice drank between 0.8 and 1.1 mg kg-1 per 24 h of MDZ, which corresponds to a pharmacological dose. Two control experiments were carried out using a similar protocol. First, we offered α1(H101R) mice a choice between water and sucrose solution. Both wild-type and mutant mice developed a strong preference for sucrose, indicating that α1(H101R) mice are not generally deficient in reward reinforcement (Supplementary Fig. 9). We also tested whether α3(H126R) mice, in which MDZ caused a normal disinhibition of DA neurons (Supplementary Fig. , would develop a preference for MDZ, which was indeed the case (Supplementary Fig. 10). Although BDZs, particularly MDZ, may enhance taste perception28, this is unlikely to influence the interpretation of these data, as several studies have shown that BDZ-mediated taste enhancement is independent of α1-containing GABAARs29, 30.

Figure 6: Oral self-administration of MDZ.

Figure 6 : Oral self-administration of MDZ. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

 

a, Protocol for behavioural experiment. b, Total consumption successively with water, sucrose and MDZ (0.005 mg ml-1) plus sucrose (4%) in wild-type mice (black) and α1(H101R) (red) mice. Note that wild-type and α1(H101R) mice drink similar amounts of liquids. c, Relative MDZ consumption in wild-type and α1(H101R) mice. d, Corresponding box plots for relative average consumption of MDZ at days (D) indicated. n = 12–18 mice in 4–6 cages; F(3;16) = 5.39. Box plot designations are as in Fig. 3d; **P < 0.01.

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Discussion

 

On the basis of our data, we propose that BDZs increase DA levels through disinhibition, similar to opioids, cannabinoids and γ-hydroxybutyrate. This disinhibition is dependent on the BDZ-binding site on α1-containing GABAARs in the VTA. The net effect of BDZs on the VTA circuit is dominated by the role of α1-containing GABAARs, which is supported by the following three observations. First, GABAAR-mediated quantal transmission is stronger in GABA neurons than in DA neurons, as evidenced by the larger charge transfer of mIPSCs (Fig. 3d). Second, GABA neurons have a higher input resistance than DA neurons17, allowing the same charge transfer to more effectively change the membrane potential of GABA neurons than DA neurons. Third, the BDZ-dependent enhancement of each IPSC on DA neurons causes little inhibition of DA neuron activity because GABA neurons fall silent and no longer generate those IPSCs. Our model could also apply to earlier work probing the effect of the GABAAR-agonist muscimol31. When administered directly into the VTA, muscimol causes an increase of DA levels in the nucleus accumbens32. This effect only occurs at low doses, which led to the conclusion that the effect is mediated indirectly on non-DA neurons33, 34. This inverse dose-dependence may be due to the fact that muscimol, unlike BDZs, is not a positive modulator but an agonist. In line with this interpretation, muscimol at high concentrations in fact inhibits DA neurons35.

 

The implication of α1 in the addictive effect of BDZs is surprising because the clinically available compound ZOL is selective for this subunit and has been claimed to carry a low risk for addiction36. However, this optimistic view contrasts with the observation that ZOL is readily self-administered37 and the clinical reality. Our data with the subunit isoform-selective compounds also show that ZOL triggers drug-evoked plasticity, and indicate that α1-sparing compounds may be promising candidates in the search for BDZs devoid of addiction liability. Because α1-containing GABAARs outside the VTA mediate other effects such as seizure control, sedation and anterograde amnesia38, α1-sparing compounds will certainly not be suitable for all indications. The dissociation between anxiolysis, mainly α2-mediated23, and addiction, however, seems possible in principle. This is particularly appealing as high anxiety levels suggest increased vulnerability for addiction39.

 

Our work unravels the molecular basis of the defining pharmacological features that BDZs share with addictive drugs, which we believe will be key for designing new BDZs with lower addiction liability. However, we note that increased levels of mesolimbic dopamine are necessary for addiction, but not sufficient on their own. Recent studies suggest that early drug-evoked plasticity in the VTA may facilitate addiction by gating more enduring forms of adaptations in target regions of the mesolimbic system, which would represent the eventual locus underlying long-term addictive behaviours40, 41. Coinciding factors of vulnerability, either in the initial events in the VTA or in subsequent events in mesolimbic targets, may ultimately explain individual variations in susceptibility to addiction, both for BDZs and for other drugs42.

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Methods Summary

 

Horizontal slices of the midbrain (250 μm) were prepared as previously described43 from C57BL/6 mice, Pitx3–GFP knock-in mice44, GAD67–GFP Δneo mice45 and α1(H101R) knock-in mice13, 24 h after i.p. or intra-VTA (mediolateral (ML): ±0.8, anteroposterior (AP): -2.4, dorsoventral (DV): -4.4 mm from bregma) injections of different BDZs. AMPAR-mediated EPSCs were recorded in the presence of d(-)-2-amino-5-phosphonovaleric acid (AP5) and picrotoxin. mIPSCs were recorded in the presence of kynurenic acid (2 mM) and tetrodotoxin (500 nM). In vivo extracellular single-unit recordings of DA neurons in the VTA (ML: -1.2, AP: -3.2, DV: -4 to 4.5 mm from the bregma) were carried out in wild-type, α1(H101R) and α3(H126R)23 knock-in mice. Drugs were delivered through the tail vein. Immunofluorescence with a guinea-pig antibody against the α1 or α3 subunit, a mouse antibody against tyrosine hydroxylase, and a rabbit antibody against enhanced GFP (eGFP) was performed as previously described10 in GAD67–GFP Δneo mice. For the oral self-administration of MDZ, mice were housed with free access to two bottles containing either MDZ in sucrose or sucrose alone. Grouped data are expressed as means ± s.e.m. For statistical comparisons the one-way analysis of variance (ANOVA), Bonferroni matched, or the paired Student’s t-tests were used. The levels of significance are indicated as follows: *P < 0.05, **P < 0.01 and ***P < 0.001.

Full methods accompany this paper.

 

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References

 

  1. Lüscher, C. & Ungless, M. A. The mechanistic classification of addictive drugs. PLoS Med. 3, e437 (2006) | Article | PubMed

  2. O’Brien, C. P. Benzodiazepine use, abuse, and dependence. J. Clin. Psychiatry 66 (suppl. 2). 28–33 (2005) | Article | PubMed | ChemPort |

  3. Saal, D., Dong, Y., Bonci, A. & Malenka, R. C. Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons. Neuron 37, 577–582 (2003) | Article | PubMed | ISI | ChemPort |

  4. Bellone, C. & Lüscher, C. Cocaine triggered AMPA receptor redistribution is reversed in vivo by mGluR-dependent long-term depression. Nature Neurosci. 9, 636–641 (2006) | Article

  5. Argilli, E., Sibley, D. R., Malenka, R. C., England, P. M. & Bonci, A. Mechanism and time course of cocaine-induced long-term potentiation in the ventral tegmental area. J. Neurosci. 28, 9092–9100 (2008) | Article | PubMed | ChemPort |

  6. Mameli, M., Balland, B., Lujan, R. & Luscher, C. Rapid synthesis and synaptic insertion of GluR2 for mGluR-LTD in the ventral tegmental area. Science 317, 530–533 (2007) | Article | PubMed | ISI | ChemPort |

  7. Heikkinen, A. E., Moykkynen, T. P. & Korpi, E. R. Long-lasting modulation of glutamatergic transmission in VTA dopamine neurons after a single dose of benzodiazepine agonists. Neuropsychopharmacol. 34, 290–298 (2009) | Article

  8. Johnson, S. W. & North, R. A. Two types of neurone in the rat ventral tegmental area and their synaptic inputs. J. Physiol. (Lond.) 450, 455–468 (1992) | PubMed | ISI | ChemPort |

  9. Sigel, E., Schaerer, M. T., Buhr, A. & Baur, R. The benzodiazepine binding pocket of recombinant α1β2γ2 γ-aminobutyric acidA receptors: relative orientation of ligands and amino acid side chains. Mol. Pharmacol. 54, 1097–1105 (1998) | PubMed | ISI | ChemPort |

  10. Fritschy, J. M. & Mohler, H. GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits. J. Comp. Neurol. 359, 154–194 (1995) | Article | PubMed | ISI | ChemPort |

  11. Okada, H., Matsushita, N., Kobayashi, K. & Kobayashi, K. Identification of GABAA receptor subunit variants in midbrain dopaminergic neurons. J. Neurochem. 89, 7–14 (2004) | Article | PubMed | ChemPort |

  12. Arendt, R. M., Greenblatt, D. J., Liebisch, D. C., Luu, M. D. & Paul, S. M. Determinants of benzodiazepine brain uptake: lipophilicity versus binding affinity. Psychopharmacology (Berl.) 93, 72–76 (1987) | Article | PubMed

  13. Rudolph, U. et al. Benzodiazepine actions mediated by specific γ-aminobutyric acidA receptor subtypes. Nature 401, 796–800 (1999) | Article | PubMed | ISI | ChemPort |

  14. Möhler, H., Benke, D., Mertens, S. & Fritschy, J. M. GABAA-receptor subtypes differing in α-subunit composition display unique pharmacological properties. Adv. Biochem. Psychopharmacol. 47, 41–53 (1992) | PubMed |

  15. McKernan, R. M. et al. Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABAA receptor alpha1 subtype. Nature Neurosci. 3, 587–592 (2000)

  16. Cameron, D. L., Wessendorf, M. W. & Williams, J. T. A subset of ventral tegmental area neurons is inhibited by dopamine, 5-hydroxytryptamine and opioids. Neuroscience 77, 155–166 (1997) | Article | PubMed | ISI | ChemPort |

  17. Margolis, E. B., Lock, H., Hjelmstad, G. O. & Fields, H. L. The ventral tegmental area revisited: is there an electrophysiological marker for dopaminergic neurons? J. Physiol. (Lond.) 577, 907–924 (2006) | Article | PubMed

  18. Hsia, A. Y., Malenka, R. C. & Nicoll, R. A. Development of excitatory circuitry in the hippocampus. J. Neurophysiol. 79, 2013–2024 (1998) | PubMed | ISI | ChemPort |

  19. Poncer, J. C., Durr, R., Gähwiler, B. H. & Thompson, S. M. Modulation of synaptic GABAA receptor function by benzodiazepines in area CA3 of rat hippocampal slice cultures. Neuropharmacology 35, 1169–1179 (1996) | Article | PubMed

  20. Long, P. et al. Nerve terminal GABAA receptors activate Ca2+/calmodulin-dependent signaling to inhibit voltage-gated Ca2+ influx and glutamate release. J. Biol. Chem. 284, 8726–8737 (2009) | Article | PubMed

  21. Barberis, A., Mozrzymas, J. W., Ortinski, P. I. & Vicini, S. Desensitization and binding properties determine distinct α1β2γ2 and α3β2γ2 GABAA receptor-channel kinetic behavior. Eur. J. Neurosci. 25, 2726–2740 (2007) | Article | PubMed

  22. Yee, B. K. et al. A schizophrenia-related sensorimotor deficit links alpha 3-containing GABAA receptors to a dopamine hyperfunction. Proc. Natl Acad. Sci. USA 102, 17154–17159 (2005) | Article | PubMed | ChemPort |

  23. Löw, K. et al. Molecular and neuronal substrate for the selective attenuation of anxiety. Science 290, 131–134 (2000) | Article | PubMed | ISI | ChemPort |

  24. Robinson, S., Smith, D. M., Mizumori, S. J. & Palmiter, R. D. Firing properties of dopamine neurons in freely moving dopamine-deficient mice: effects of dopamine receptor activation and anesthesia. Proc. Natl Acad. Sci. USA 101, 13329–13334 (2004) | Article | PubMed

  25. Hyland, B. I., Reynolds, J. N., Hay, J., Perk, C. G. & Miller, R. Firing modes of midbrain dopamine cells in the freely moving rat. Neuroscience 114, 475–492 (2002) | Article | PubMed | ISI | ChemPort |

  26. Pilotto, R., Singer, G. & Overstreet, D. Self-injection of diazepam in naive rats: effects of dose, schedule and blockade of different receptors. Psychopharmacology (Berl.) 84, 174–177 (1984) | Article | PubMed

  27. Fuchs, V., Burbes, E. & Coper, H. The influence of haloperidol and aminooxyacetic acid on etonitazene, alcohol, diazepam and barbital consumption. Drug Alcohol Depend. 14, 179–186 (1984) | Article | PubMed

  28. Berridge, K. C. & Pecina, S. Benzodiazepines, appetite, and taste palatability. Neurosci. Biobehav. Rev. 19, 121–131 (1995) | Article | PubMed

  29. Yerbury, R. E. & Cooper, S. J. Novel benzodiazepine receptor ligands: palatable food intake following zolpidem, CGS 17867A, or Ro23–0364, in the rat. Pharmacol. Biochem. Behav. 33, 303–307 (1989) | Article | PubMed

  30. Morris, H. V., Nilsson, S., Dixon, C. I., Stephens, D. N. & Clifton, P. G. α1- and α2-containing GABAA receptor modulation is not necessary for benzodiazepine-induced hyperphagia. Appetite 52, 675–683 (2009) | Article | PubMed

  31. Kalivas, P. W., Duffy, P. & Eberhardt, H. Modulation of A10 dopamine neurons by γ-aminobutyric acid agonists. J. Pharmacol. Exp. Ther. 253, 858–866 (1990) | PubMed | ISI | ChemPort |

  32. Xi, Z. X. & Stein, E. A. Nucleus accumbens dopamine release modulation by mesolimbic GABAA receptors-an in vivo electrochemical study. Brain Res. 798, 156–165 (1998) | Article | PubMed | ISI | ChemPort |

  33. Doherty, M. & Gratton, A. Differential involvement of ventral tegmental GABAA and GABAB receptors in the regulation of the nucleus accumbens dopamine response to stress. Brain Res. 1150, 62–68 (2007) | Article | PubMed

  34. Grace, A. A. & Bunney, B. S. Paradoxical GABA excitation of nigral dopaminergic cells: indirect mediation through reticulata inhibitory neurons. Eur. J. Pharmacol. 59, 211–218 (1979) | Article | PubMed | ISI | ChemPort |

  35. Klitenick, M. A., DeWitte, P. & Kalivas, P. W. Regulation of somatodendritic dopamine release in the ventral tegmental area by opioids and GABA: an in vivo microdialysis study. J. Neurosci. 12, 2623–2632 (1992) | PubMed | ISI | ChemPort |

  36. Soyka, M., Bottlender, R. & Moller, H. J. Epidemiological evidence for a low abuse potential of zolpidem. Pharmacopsychiatry 33, 138–141 (2000) | Article | PubMed

  37. Rowlett, J. K., Platt, D. M., Lelas, S., Atack, J. R. & Dawson, G. R. Different GABAA receptor subtypes mediate the anxiolytic, abuse-related, and motor effects of benzodiazepine-like drugs in primates. Proc. Natl Acad. Sci. USA 102, 915–920 (2005) | Article | PubMed | ChemPort |

  38. Rudolph, U. & Möhler, H. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 6, 18–23 (2005) | Article | PubMed

  39. Koob, G. F. Dynamics of neuronal circuits in addiction: reward, antireward, and emotional memory. Pharmacopsychiatry 42 (suppl. 1). S32–S41 (2009) | Article | PubMed

  40. Lüscher, C. & Bellone, C. Cocaine-evoked synaptic plasticity: a key to addiction? Nature Neurosci. 11, 737–738 (2008) | Article

  41. Mameli, M. et al. Cocaine-evoked synaptic plasticity: persistence in the VTA triggers adaptations in the NAc. Nature Neurosci. 12, 1036–1041 (2009) | Article

  42. Redish, A. D., Jensen, S. & Johnson, A. A unified framework for addiction: vulnerabilities in the decision process. Behav. Brain Sci. 31, 415–437 (2008) | PubMed |

  43. Labouèbe, G. et al. RGS2 modulates coupling between GABAB receptors and GIRK channels in dopamine neurons of the ventral tegmental area. Nature Neurosci. 10, 1559–1568 (2007) | Article

  44. Zhao, S. et al. Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons. Eur. J. Neurosci. 19, 1133–1140 (2004) | Article | PubMed | ISI

  45. Tamamaki, N. et al. Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60–79 (2003) | Article | PubMed | ChemPort |

  46. Paxinos, G. & Franklin, K. B. J. The Mouse Brain in Stereotaxic Coordinates (Elsevier, 2004)

  47. Grace, A. A. & Bunney, B. S. Intracellular and extracellular electrophysiology of nigral dopaminergic neurons—1. Identification and characterization. Neuroscience 10, 301–315 (1983) | Article | PubMed | ISI | ChemPort |

  48. Ungless, M. A., Magill, P. J. & Bolam, J. P. Uniform inhibition of dopamine neurons in the ventral tegmental area by aversive stimuli. Science 303, 2040–2042 (2004) | Article | PubMed | ISI | ChemPort |

 

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Supplementary Information

 

Supplementary information accompanies this paper.

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Acknowledgements

 

We thank members of the Lüscher laboratory as well as M. Frerking, M. Serafin and H. Möhler for critical reading of the manuscript. Y. Yanagawa provided the GAD67–GFP Δneo mouse line, and we thank K. A. Miczek and H. U. Zeilhofer for help with the GABAA mutant mouse lines. This work is supported by the National Institute on Drug Abuse (NIDA; DA019022; C.L., P. Slesinger), the Swiss National Science Foundation, the Swiss Initiative in Systems Biology (Neurochoice) and the European Commission Coordination Action ENINET (LSHM-CT-2005-19063). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIDA or the National Institutes of Health.

 

Author Contributions K.R.T. carried out all in vitro electrophysiology experiments. M.B., G.L. and C.Y. contributed equally to the in vivo recordings. K.R.T. and C.C. performed the behavioural experiments. J.-M.F. carried out the immunohistochemistry. U.R. generated the mutant mice. C.L. designed the study and wrote the manuscript with the help of all authors.

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Competing interests statement

 

The authors declare no competing financial interests.

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Online Methods

Animals

 

Animals used were 2–3-week-old (in vitro electrophysiology) and 20–32-week-old (in vivo electrophysiology/behaviour) wild-type C57BL/6 mice, Pitx3–GFP knock-in mice44, GAD67–GFP Δneo mice45, α1(H101R) knock-in mice13 and α3(H126R) knock-in mice23. All procedures were approved by the local ethics committee as well as the cantonal authorities of Geneva.

In vitro electrophysiology

 

Horizontal slices (250-μm thick) of the midbrain were prepared as described previously43. Slices were kept in ACSF containing (in mM) 119 NaCl, 2.5 KCl, 1.3 MgCl2, 2.5 CaCl2, 1.0 NaH2PO4, 26.2 NaHCO3 and 11 glucose, and bubbled with 95% O2 and 5% CO2. The whole-cell voltage-clamp recording technique was used (31–33 °C, 2 ml min-1, submerged slices) to measure synaptic responses of DA neurons, mIPSCs and holding currents of DA or GABA neurons of the VTA. The holding potential was -60 mV and the access resistance was monitored by a hyperpolarizing step to -90 mV with each sweep every 10 s. Experiments were terminated if the access resistance varied more than 20%. Synaptic currents were evoked by stimuli (0.1 ms) at 0.05 Hz through bipolar stainless steel electrodes positioned rostral to the VTA. When EPSCs were recorded, the internal solution was composed of (in mM) 130 CsCl, 4 NaCl, 2 MgCl2, 1.1 EGTA, 5 HEPES, 2 Na2ATP, 5 Na2-creatine-phosphate, 0.6 Na3GTP and 0.1 spermine, whereas for mIPSCs the internal solution used contained 30 potassium-gluconate, 100 KCl, 4 MgCl2, 1.1 EGTA, 5 HEPES, 3.4 Na2ATP, 10 creatine-phosphate and 0.1 Na3GTP. Currents were amplified ( Multiclamp 700A, Molecular Devices), filtered at 1 kHz and digitized at 5 kHz ( National Instruments Board PCI-MIO-16E4, Igor, WaveMetrics). As the liquid junction potential was -3 mV, traces were not corrected. Recordings of EPSCs were carried out in the presence of picrotoxin (100 μM) and AP5 (50 μM). The rectification index was calculated by dividing the amplitude of the AMPAR-EPSCs measured at -65 mV by the amplitude at +35 mV. sIPSCs were recorded with continuous bath-application of kynuric acid (2 mM), and tetrodotoxin (500 nM) was added to measure mIPSCs. When sIPSCs were recorded (Fig. 4), the bath-applied ACSF contained a Ca2+/Mg2+ ratio of 3–6. The goal was to increase the number of interneuronal spikes while interfering with the GABAergic output per spike as little as possible. The multiplicity factor was calculated following the protocol described previously18. At the end of each experiment, picrotoxin (100 μM) was bath-applied to verify that the recorded current was mediated by GABAARs.

In vivo electrophysiology

 

Mice were initially anaesthetized with 4% chloral hydrate (480 mg kg-1, i.p.), and supplemented each hour with a lower dose (120 mg kg-1 i.p.) to maintain optimal anaesthesia throughout the experiment. Animals were positioned in a stereotaxic frame (MyNeurolab) and body temperature was maintained at 36–37 °C using a feedback-controlled heating pad (Harvard Apparatus). An incision was made in the midline to expose the skull. A burr hole was unilaterally drilled above the VTA (AP: -3.0 to 3.4, ML: -1.1 to 1.4, DV: -4 to 4.5 mm from the bregma)46, and the dura was carefully retracted. Electrodes were broken back to give a final tip diameter of 1–2 μm and filled with 2% Chicago Sky Blue dye in 0.5 M sodium-acetate. All electrodes had impedance of 15–25 MΩ. They were angled by 10° from the vertical, slowly lowered through the burr hole with a micro drive (Luigs Neumann) and positioned in the VTA. All electrode descents within a single animal were a minimum of 100 μm apart. A reference electrode was placed in the subcutaneous tissue. Electrical signals were AC-coupled, amplified (Neurodata), and monitored in real time using an audiomonitor (homemade). Signals were filtered on-line ( Humbug, Quest scientific) and digitized at 20 kHz (for waveform analysis) or 5 kHz ( Igor, WaveMetrics). The bandpass filter was set between 0.3 and 5 kHz. Extracellular identification of VTA neurons was on the basis of their location as well as on their established electrophysiological properties (DA neurons: biphasic action potential of more than 1.1 ms duration, firing frequency of 0.5–7 Hz and spike height accommodation during bursts)47, 48. In addition, we discriminated between the two populations using an aversive electrical footshock and response to morphine. The drugs were injected through the tail vein using a cannula. After completion of recordings, Chicago Sky Blue dye was deposited by iontophoresis (-15 μA, 15 min) to mark the position of the final recording site. At the end of the experiment, the brain was kept at -20 °C in a solution of methyl butane. Fifty-micrometre thick coronal sections were cut on a cryostat, stained with luxol fast blue/cresyl violet and the recording site was verified by light microscopy.

Stereotaxic injection

 

Wild-type and α1(H101R) mice were anaesthetized with ketamine (100 mg kg-1) and xylazine (10 mg kg-1). The animal was then placed in a stereotaxic frame (MyNeurolab). The VTA coordinates were ML: ±0.8, AP: -2.4, DV: -4.4 mm from bregma, and verified with ink injections. Five microlitres of an 8 mg ml-1 MDZ solution or 5 μl ACSF were injected bilaterally over 10 min. The animal was sutured and recovered for 24 h until in vitro recordings were made.

Immunohistochemistry

 

GAD67–GFP Δneo mice were anaesthetized with nembutal (50 mg kg-1) and perfused transcardially with 4% paraformaldehyde in phosphate buffer. The brain was extracted and post-fixed for 3 h, cryoprotected in 30% sucrose in PBS, frozen, and cut at 40 μm with a sliding microtome. Triple immunofluorescence with guinea-pig antibody against the α1 or α3 subunit, a mouse antibody against tyrosine hydroxylase, and a rabbit antibody against eGFP was performed as previously described10 in perfusion-fixed transverse sections from the brain of GAD67–GFP Δneo mice. Images were taken with a laser scanning confocal microscope using a ×20 (numerical aperture (NA) 0. or a ×63 (NA 1.4) objective, using sequential acquisition of separate channels to avoid cross-talk. The fraction of neurons single- and double-labelled for these markers was assessed pair-wise (for example, α1/TH or α3/GAD67–GFP) in four equally spaced sections through the VTA per mouse (n = 4) and expressed as a percentage of the total number of cells counted.

Oral self-administration

 

Mice were habituated to handling for 1 week and housed with free access to two 450-ml plastic bottles in their home cage. Two days before the test, 4% sucrose was added to both bottles. During the test mice had access to bottles containing either MDZ (0.005 mg ml-1) in sucrose or sucrose alone. For the sucrose preference experiment in α1(H101R) mice, sucrose was compared against water. In cases where mice spontaneously preferred one bottle to another during the pretest phase, MDZ was always added to the least-preferred bottle during the test phase. To determine MDZ preference, the relative consumption of MDZ solution to the control solution was calculated.

Drugs

 

MDZ, diazepam, flunitrazepam, flumazenil, ZOL and L-838 417 were supplied by Tocris, and morphine-HCl by the pharmacy of the University Hospital of Geneva. Drugs were dissolved in saline for i.p. and i.v. injections, in ACSF for intra-VTA injections, and in dimethyl sulphoxide (DMSO) for bath-applications. The final DMSO concentration was 0.1%.

Statistics

 

Grouped data are expressed as means ± s.e.m. or box-plots (median, interquartile, and 90th and 10th percentiles). For statistical comparisons the one-way ANOVA, Bonferroni matched, or the paired Student’s t-tests were used. The levels of significance are: *P < 0.05, **P < 0.01 and ** P < 0.001. The Kolmogorov–Smirnov test was used to compare cumulative probability plots.

Interesting Daily Mail article. There but for the grace of God go I. In the 70's valium were handed out in buckets and just about used for lolly scrambles. Want three months supply? - no problemo and have some tryptonol too. Run out, just ask your neighbour friend or workmate for a couple to tide you over. I saw the light and bailed out but many were caught and still are. What a crime!

This makes me so sad.

 

Some of you know that my mom died 8 years ago from cancer. Since my journey with benzos, I've been able to look back and I realize that my mom was almost certainly on benzos when I was growing up.  I can distinctly remember her freaking out over the smallest things, and saying she needed her medicine, and then taking a tiny blue pill.  I now realize that it was probably Valium.  I don't know this for a fact, but the more I think back to my childhood/adolescent years and the way she acted, the more certain I am.

 

It truly saddens me that so many people are accidental addicts of these drugs. Makes you wonder about people in their 60's and 70's who have been diagnosed with Alzheimers.  Is it truly Alz or is it benzos?

 

Thank goodness for people like Colin, Professor Ashton and others who are now educating us on the dangers of these nasty drugs.

Thanks Beeper. I took a read at it.

 

Missy, sorry to hear about your mother.

 

It truly saddens me that so many people are accidental addicts of these drugs. Makes you wonder about people in their 60's and 70's who have been diagnosed with Alzheimers.  Is it truly Alz or is it benzos?

 

You bring up a good point. My grandfather has been on Valium for years. He was taken off the Valium suddenly with very little withdrawal, but eventually he started feeling memory loss and so on.. so sadly, the doctor put him on Xanax. He has been dx with Alzheimers. I told my mother I think it's the Benzo's. She thinks it could be as well.. he's in his 80's and he will not come off the Xanax.

 

It makes one wonder.

 

S#

 

 

Thanks for the post beeper.  We can only hope that increasing awareness helps. 

 

Draftsman

Thanks for posting this Beeper. Very sad but I hope it helps awareness as well.