Currently, the structures responsible for the onset, propagation, and cessation of withdrawal convulsions are not known. The present studies are the first to show that the clSNr plays a crucial role in barbiturate and ethanol withdrawal and that intrinsic neurons rather than fibers of passage are essential to its role in withdrawal. These findings demonstrate that the clSNr plays a crucial role within a brain circuit involved in withdrawal from PB and ethanol and potentially other drugs.
Recently, we identified the SNr as a brain region that exhibits genotype-dependent neuronal activation associated with ethanol withdrawal [22
]. Although electrolytic lesions of the clSNr implicated this structure in ethanol withdrawal [22
], they also damage fibers of passage [23
]. In contrast, ibotenic acid lesions produce little or no damage to fibers of passage at appropriate doses [23
]. Consistent with these reports, we found in the present studies that necrotic tissue was frequently observed at electrolytic lesion sites, but was not detected at the chemical lesion sites. Our results demonstrate that the reduction in PB withdrawal severity resulting from bilateral chemical lesions of the clSNr was comparable in magnitude to that resulting from bilateral electrolytic lesions of this region. These results demonstrate that intrinsic clSNr cells play a critical role in PB withdrawal.
Ethanol withdrawal was also substantially attenuated by bilateral chemical lesions of the clSNr, indicating that intrinsic cells are crucial to the role of the clSNr on both ethanol and PB withdrawal. The degree to which ethanol withdrawal was reduced by ibotenic acid lesions (~60%) may be less than we reported previously after bilateral electrolytic lesions (which reduced ethanol withdrawal by 65–95% using acute and repeated ethanol exposure models [22
]. A potentially smaller effect of chemical lesions might be influenced by the more limited extent of the chemical lesions, which was apparent in the present studies. It might also reflect potential differences in injury to regions remote to the target sites when different lesion methods are used [24
]. Finally, a potentially smaller effect of chemical lesions could also suggest that, in addition to intrinsic clSNr cells, fibers passing through the clSNr that terminate in other brain regions may also contribute to ethanol withdrawal. However, this was not apparent for PB withdrawal.
Our results confirm that the clSNr is critical for both PB and alcohol withdrawal, but additional brains regions are certainly involved in withdrawal [40
]. The SNr receives afferents from other basal ganglia regions including the striatum and subthalamic nucleus [43
], which are also implicated in CNS hyperexcitability phenotypes and might plausibly play a role in SH drug withdrawal. Interestingly, bilateral electrolytic lesions of the striatal ventral caudate putamen exacerbate ethanol withdrawal [44
], while electrolytic lesions of the subthalamic nucleus do not affect ethanol withdrawal convulsions [22
]. The deep layers of the superior colliculus (DLSC) receive a significant projection from the SNr as well as multimodal sensory information including visual, acoustic, somato-vesticular inputs [45
]. SNr projections to the DLSC may gate the sensorimotor association and influence the motor centers for expression of convulsions, such as motor neurons in the nucleus reticularis pontis oralis [46
]. The mesopontine tegmentum receives projections from the SNr as well as other basal ganglia regions [47
], and microinjection of PB into this brain area depresses CNS activity [48
], so it is plausible that rebound hyperexcitability in this region might contribute to withdrawal from PB and other SH drugs. Much future work will be needed to fully elucidate the neural networks modulating the SH drug withdrawal syndrome.
The acute actions of both ethanol and barbiturates include the enhancement of inhibitory transmission mediated by GABA, including effects on GABAA
receptors and GABA release in many brain regions [49
]. Withdrawal from these drugs represents the manifestation of physiological compensation to their effects and is thought to involve changes in GABAergic transmission [53
]. It is therefore interesting, though not surprising, that the clSNr lesions reduced ethanol and PB withdrawal convulsions, but did not affect convulsions in response to PTZ, which blocks GABAA
receptor-mediated transmission [37
]. Consistent with this observation, mice congenic for a QTL on chromosome 4 QTL affecting PB and ethanol withdrawal do not differ in PTZ-enhanced HICs [55
]. This may suggest that the chromosome 4 QTL affects GABAergic ‘tone’ rather than GABAA
receptor function per se
. In the SNr, activation of presynaptic GABAB
receptors reduces GABA release and suppresses the frequency of synaptic currents mediated by postsynaptic GABAA
]. Also in the SNr, 5-HT2C
receptor activation increases the frequency and amplitude of these GABAA
receptor mediated synaptic currents, suggesting an effect on GABA release [56
]. Both GABAB
receptors physically associate with the multi-PDZ domain protein (MPDZ, also called MUPP1 [57
]) encoded by Mpdz
, a quantitative trait gene for the ethanol and PB withdrawal QTLs on chromosome 4 [10
]. PDZ domain proteins affect the function/expression of proteins with which they physically associate [59
], so is plausible that MPDZ affects GABAB
receptor function/expression and thereby affects GABAergic tone and withdrawal. Indeed, preliminary analyses suggest that central administration of baclofen exacerbates withdrawal in mice (Kruse L and Buck K, unpublished data), and that microinjection of a preferential 5-HT2C
receptor agonist into the clSNr attenuates withdrawal severity (Chen G and Buck K, unpublished data). Diminished 5-HT2C
receptor expression may be functionally related to the increased susceptibility to respiratory arrest induced by generalized convulsive seizures in DBA/2 compared to C57BL/6J mice [60
]. Future studies will be needed to test the hypothesis that MPDZ affects GABAB
receptor function in the SNr and other brain regions and thereby affects withdrawal.
Our findings advance our understanding of the neural determination of drug withdrawal. To date, we have assessed the impact of SNr lesions on two drugs, so it remains to be determined to what extent our results generalize to additional drugs. Second, this study focuses on withdrawal HICs. While many signs of the withdrawal syndrome are genetically correlated with HIC severity (i.e., tremors, hypoactivity, emotionality; [3
], others are not [i.e., tail stiffness; 18
]. Future studies will be needed to determine to what degree the clSNr and associated brain circuitry affect other withdrawal signs. Despite these limitations, the current study demonstrates that the clSNr is crucially involved in barbiturate and ethanol withdrawal.