The Edinger-Westphal-lateral septum urocortin pathway and its relationship to alcohol consumption

Identifying and characterizing brain regions regulating alcohol consumption is beneficial for understanding the mechanisms of alcoholism. To this aim, we first identified brain regions changing in expression of the inducible transcription factor c-Fos in the alcohol-preferring C57BL/6J (B6) and alcohol-avoiding DBA/2J (D2) mice after ethanol consumption. Drinking a 5% ethanol/10% sucrose solution in a 30 min limited access procedure led to induction of c-Fos immunoreactivity in urocortin (Ucn)-positive cells of the Edinger-Westphal nucleus (EW), suppression of c-Fos immunoreactivity in the dorsal portion of the lateral septum (LS) of both strains of mice, and strain-specific suppression in the intermediate portion of the LS and the CA3 hippocampal region. Because the EW sends Ucn projections to the LS, and B6 and D2 mice differ dramatically in EW Ucn expression, we further analyzed the Ucn EW–LS pathway using several genetic approaches. We find that D2 mice have higher numbers of Ucn-immunoreactive processes than B6 mice in the LS and that consumption of ethanol/sucrose in the F2 offspring of a B6D2 intercross positively correlates with Ucn immunoreactivity in the EW and negatively correlates with Ucn immunoreactivity in the LS. In agreement with these findings, we find that alcohol-avoiding male B6.D2Alcp1 line 2.2 congenic mice have lower Ucn immunoreactivity in the EW than male B6.B6 mice. Finally, we also find that HAP mice, selectively bred for high alcohol preference, have higher Ucn immunoreactivity in EW, than LAP mice, selectively bred for low alcohol preference. Taken together, these studies provide substantial evidence for involvement of the EW–LS Ucn pathway in alcohol consumption

Two Functionally Distinct Serotonergic Projections into Hippocampus

Hippocampus receives dense serotonergic input specifically from raphe nuclei. However, what information is carried by this input and its impact on behavior has not been fully elucidated. Here we used in vivo two-photon imaging of activity of hippocampal median raphe projection fibers in behaving male and female mice and identified two distinct populations: one linked to reward delivery and the other to locomotion. Local optogenetic manipulation of these fibers confirmed a functional role for these projections in the modulation of reward-induced behavior. The diverse function of serotonergic inputs suggests a key role in integrating locomotion and reward information into the hippocampal CA1

Temporal Differentiation of pH-Dependent Capacitive Current from Dopamine

Voltammetric recording of dopamine (DA) with fast-scan cyclic voltammetry (FSCV) on carbon fiber microelectrodes have been widely used, because of its high sensitivity to dopamine. However, since an electric double layer on a carbon fiber surface in a physiological ionic solution behaves as a capacitor, fast voltage manipulation in FSCV induces large capacitive current. The faradic current from oxidation/reduction of target chemicals must be extracted from this large background current. It is known that ionic shifts, including H⁺, influence this capacitance, and pH shift can cause confounding influences on the FSCV recordings within a wide range of voltage. Besides FSCV with a triangular waveform, we have been using rectangular pulse voltammetry (RPV) for dopamine detection in the brain. In this method, the onset of a single pulse causes a large capacitive current, but unlike FSCV, the capacitive current is restricted to a narrow temporal window of just after pulse onset (<5 ms). In contrast, the peak of faradic current from dopamine oxidation occurs after a delay of more than a few milliseconds. Taking advantage of the temporal difference, we show that RPV could distinguish dopamine from pH shifts clearly and easily. In addition, the early onset current was useful to evaluate pH shifts. The narrow voltage window of our RPV pulse allowed a clear differentiation of dopamine and serotonin (5-HT), as we have shown previously. Additional recording with RPV, alongside FSCV, would improve identification of chemicals such as dopamine, pH, and 5-HT

Lesions of the Edinger–Westphal nucleus in C57BL/6J mice disrupt ethanol-induced hypothermia and ethanol consumption

Abstract The Edinger-Westphal nucleus (EW) is a brain region that has recently been implicated as an important novel neural target for ethanol. Thus, the EW is the only brain region consistently showing elevated c-Fos expression following both voluntary and involuntary ethanol administration. Ethanol-induced c-Fos expression in the EW has been shown to occur in urocortin I-positive neurons. Moreover, previous reports using several genetic models have demonstrated that differences in the EW urocortin I system are correlated with ethanol-mediated behaviours such as ethanol-induced hypothermia and ethanol consumption. The aim of this study was to confirm these relationships using a more direct strategy. Thus, ethanol responses were measured following electrolytic lesions of the EW in male C57BL ⁄ 6J mice. Both EW-lesioned and sham-operated animals were tested for several ethanol sensitivity measures and ethanol consumption in a two-bottle choice test. The results show that lesions of the EW significantly disrupted ethanol-induced hypothermia, while having no effect on pupillary dilation, locomotor activity or ethanol-induced sedation. In addition, EW-lesioned animals showed significantly lower ethanol preference and total ethanol dose consumed in the two-bottle choice test. EW-lesioned animals also consumed less sucrose than sham-operated animals, but did not have altered preferences for sucrose or quinine in a two-bottle choice test. These data support previously observed genetic correlations between EW urocortin I expression and both ethanol-induced hypothermia and ethanol consumption. Taken together, the findings suggest that the EW may function as a sensor for ethanol, which can influence ethanol consumption and preference

Data from: Reward-induced phasic dopamine release in the monkey ventral striatum and putamen

In-vivo voltammetry has successfully been used to detect dopamine release in rodent brains, but its application to monkeys has been limited. We have previously detected dopamine release in the caudate of behaving Japanese monkeys using diamond microelectrodes (Yoshimi 2011); however it is not known whether the release pattern is the same in various areas of the forebrain. Recent studies have suggested variations in the dopaminergic projections to forebrain areas. In the present study, we attempted simultaneous recording at two locations in the striatum, using fast-scan cyclic voltammetry (FSCV) on carbon fibers, which has been widely used in rodents. Responses to unpredicted food and liquid rewards were detected repeatedly. The response to the liquid reward after conditioned stimuli was enhanced after switching the prediction cue. These characteristics were generally similar between the ventral striatum and the putamen. Overall, the technical application of FSCV recording in multiple locations was successful in behaving primates, and further voltammetric recordings in multiple locations will expand our knowledge of dopamine reward responses

Reward-Induced Phasic Dopamine Release in the Monkey Ventral Striatum and Putamen

<div><p>In-vivo voltammetry has successfully been used to detect dopamine release in rodent brains, but its application to monkeys has been limited. We have previously detected dopamine release in the caudate of behaving Japanese monkeys using diamond microelectrodes (Yoshimi 2011); however it is not known whether the release pattern is the same in various areas of the forebrain. Recent studies have suggested variations in the dopaminergic projections to forebrain areas. In the present study, we attempted simultaneous recording at two locations in the striatum, using fast-scan cyclic voltammetry (FSCV) on carbon fibers, which has been widely used in rodents. Responses to unpredicted food and liquid rewards were detected repeatedly. The response to the liquid reward after conditioned stimuli was enhanced after switching the prediction cue. These characteristics were generally similar between the ventral striatum and the putamen. Overall, the technical application of FSCV recording in multiple locations was successful in behaving primates, and further voltammetric recordings in multiple locations will expand our knowledge of dopamine reward responses.</p></div

Electrode positioning.

<p>(A) Carbon fiber microelectrodes. Left: First design of microelectrode coated by a silica tube with 0.09 mm diameter, used in part of the early MFB stimulation experiments. Right: Microelectrode coated by a glass capillary, used for the most recordings in this study. Scale bar = 0.5 mm. Note the diameter next to the carbon fiber is thinner in the glass design (right). (B) Schematic drawing of the guide cannula (blue) (OD 0.7 mm, ID 0.42 mm). Left: An inset was placed to protect the inside of the tube when inserting into the brain. Right: The inset was replaced by the elongated carbon fiber microelectrode with a shaft of silica tube of 0.35 mm diameter. (C) Grid with vertical holes of 0.7 mm diameter and two manual-drive micromanipulators. (D) Coronal section immunostained with tyrosine hydroxylase (TH) showing the position of the striatum and MFB. (E) Schematic drawing of the grid on the monkey’s head. The electrode path is shown on the left MRI image. Note the electrode chamber on the skull, as visualized by filling with 2% agar.</p

Examples of handed food reward responses in two monkeys (S and C).

<p>Simultaneous recording in ventral striatum (top) and putamen (bottom). The recorded positions are indicated on the MRI images. A small soft biscuit (Tamago-boro) was manually delivered by the examiner. The motion of the examiner turning back and picking up a biscuit from a table and putting it to the mouth of the monkey, occurred within 2 s. The data were aligned to the moment the biscuit touched the mouth. Note there is no physical/ingestional event before time 0, but only social observation. Vertical lines indicate s.e.m. S6 n = 24–25, C4 n = 46</p

Representative example of juice reward task (Exp C4, putamen).

<p>(A) Color plot indicating the averaged differential voltammogram of free (left), CS+ (middle) and CS- (right) trials. Abscissa indicates time from juice delivery/CS offset. The ordinate indicates the time from the triangular pulse onset, from the bottom to the top. Note onset of positive response after the juice delivery in free trials (left). Similar response started at -1.5s in the CS+ and CS- trials (middle and right). (B) The current change at the dopamine oxidation peak potential (2.4 ms from the pulse onset). Free (black), CS+ (red with circle) and CS- (blue) trials. Average of 19 to 36 trials are shown with vertical bars indicating s.e.m. Inset on the right shows voltammogram 0.5 s after the free juice onset from averaged differential current (A left). (C) Dopamine-like component extracted by PCR analysis of the same data. (D) In-vivo template waveforms of dopamine and pH extracted from the MFB stimulation experiments as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130443#pone.0130443.g002" target="_blank">Fig 2</a>. (E) Same as C, but in-vitro template was used instead of in-vivo template. (F) Dopamine and pH waveforms obtained in-vitro, used for the analysis in E. Note the result value (ordinate) from in vivo template (D) is originally given as peak current (C, nA), while concentration (nM) value is directly given (E) from in vitro template (F). Vertical lines in B, C and E indicate onset of timing cue (-2.5s) and onset of CS (-1.5s)</p

Reversed CS trial (Exp C6, ventral striatum).

<p>Additional example of FSCV recording from the ventral striatum. (A and B) Ordinary CS condition as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130443#pone.0130443.g003" target="_blank">Fig 3</a>. (B) Average of PCR result of 18 to 37 trials are shown with vertical bars indicating s.e.m. Inset on the right shows voltammogram 0.9 s after the free juice onset from averaged differential current (A left). (C and D) Reversed session in which juice was delivered following the ordinary CS- (blue square), instead of the ordinary CS+ (red circle). Average of 10 to 11 trials. Note that during the reversed session (C and D), a large response to juice delivery followed the ordinary CS-. Inset on the right in D shows voltammogram 0.8 s after the juice onset following CS- from averaged differential current (C right). A positive dopamine-like response was also induced by the timing cue (-2.5 to -1.5 s). (E) The direct comparison between actual juice delivery following ordinal CS+ and CS- (*: p <0.01, by t-test (two-tailed)). Vertical lines in B, D and E indicate onset of timing cue (-2.5s) and onset of CS (-1.5s)</p