Fig 1.
Photomicrograph of a typical OTH brain section.
Photomicrograph shows retrogradely labeled PGi neurons in the rostra1 ventral medulla of a rat which received intra-LC microinjection of HRP (A, left side). Insets illustrate high magnification of LC and PGi regions. Following a single pulse electrical stimulation of PGi, an evoked EPSC was recorded from LC neuron (A, right side). The schematic representation of cutting stage orientation for preparation of OTH brain slices. The tissue block containing LC and PGi were mounted on the wedge-shaped agarose gel with an angle of 50°. The red dashed line shows the cutting direction (B). Picture shows the blade direction and the way through which tissue block was mounted on cutting stage (C). OTH: oblique to horizontal; LC: locus coeruleus; PGi: paragigantocellularis; HRP: horseradish peroxidase; EPSC: excitatory post synaptic current.
Fig 2.
SDR and RMP of LC neurons in naïve (non-dependent) rats.
Representative traces show the spontaneous discharge activity of three LC neurons before and 5 min after naloxone superfusion (1 μM) in HZ (A), OTH-cut (B) and OTH (C) brain slices. Histograms (A–C) indicate the mean RMP and frequency of SDR recorded before and after naloxone application. Note that naloxone did not alter the SDR and RMP of LC neurons in naïve rats. The dashed line represents the RMP. Summary data showing the mean SDR and RMP in LC neurons of all three slice forms in naïve rats, which were not significantly different among themselves (D). Data are expressed as mean ± SEM, n = 8 in each type of brain slice. Data were analyzed using paired Student’s t-test (A–C) and one-way ANOVA followed by Tukey’s post hoc test (D). SDR: spontaneous discharge rates; RMP: resting membrane potential; NLX: naloxone; LC: locus coeruleus; HZ: horizontal; OTH-cut: oblique to horizontal-cut; OTH: oblique to horizontal.
Fig 3.
Effect of acute morphine application on SDR and RMP of LC neurons in naïve rats.
Representative traces show the RMP and SDR of three LC neurons before and 5 min after naloxone superfusion (1 μM) in HZ (A), OTH-cut (B) and OTH (C) brain slices (slices taken from naïve animals were incubated with 5 μM morphine for 60 to 90 min before naloxone application). Histograms (A–C) indicate the mean RMP and frequency of SDR recorded before and after naloxone application. Acute incubation of brain slices with 5 μM morphine led to significant decrement in RMP and suppression of the SDR of LC neurons in non-dependent animals. Note that naloxone returned SDR and RMP values to the baseline levels in naïve animals. It should be noted that the net effect of naloxone on RMP and SDR was similar among the three forms of brain slices (D). The dashed line represents the RMP after naloxone superfusion. Data are expressed as mean ± SEM, n = 8 in each type of brain slice, *** P < 0.001, compared to before naloxone application. Data were analyzed using paired Student’s t-test (A–C) and one-way ANOVA followed by Tukey’s post hoc test (D). RMP: resting membrane potential; SDR: spontaneous discharge rates; NLX: naloxone; LC: locus coeruleus; HZ: horizontal; OTH-cut: oblique to horizontal-cut; OTH: oblique to horizontal.
Fig 4.
Effect of naloxone on SDR and RMP of LC neurons in morphine-dependent rats.
Representative traces show the spontaneous discharge activity and the RMP of three LC neurons before and 5 min after naloxone superfusion (1 μM) in HZ (A), OTH-cut (B) and OTH (C) brain slices. The slices were bathed in 5 μM morphine. Histograms indicate the mean RMP and frequency of SDR recorded before and after naloxone application. Naloxone significantly increased the SDR and RMP of LC neurons in all form of brain slices. It should be noted that the net (not the mere) effect of naloxone on RMP and SDR was significantly higher in OTH brain slices than those of HZ and OTH-cut preparations taken from morphine dependent rats (D). Data are expressed as mean ± SEM, n = 8 in each type of brain slice, * P < 0.05, ** P < 0.01 and *** P < 0.001 compare to HZ and OTH-cut. Data were analyzed using paired Student’s t-test (A–C) and one-way ANOVA followed by Tukey’s post hoc test (D). RMP: resting membrane potential; SDR: spontaneous discharge rates; NLX: naloxone; LC: locus coeruleus; HZ: horizontal; OTH-cut: oblique to horizontal-cut; OTH: oblique to horizontal.
Fig 5.
Effect of kynurenic acid on LC neuronal activity following morphine withdrawal in morphine-dependent rats.
Summary data showing the effect of kynurenic acid (500 μM) on the SDR (A: total and C: net) and RMP (B: total and D: net) of LC neurons following opiate withdrawal in all three slice forms taken from morphine treated rats. Kynurenic acid application in HZ and OTH-cut brain slices did not significantly affect the net increased SDR and RMP values following withdrawal-induced hyperactivity of LC neurons. Also, Treatment of OTH brain slices with kynurenic acid deceases the observed increment in LC neuronal SDR and RMP in comparison to HZ and OTH-cut brain slices. Data are expressed as mean ± SEM, n = 6–8 in each type of brain slice, * P < 0.05 versus LC neuronal SDR and RMP from OTH brain slices without kynurenic acid application. Data were analyzed using One-way ANOVA followed by Tukey’s post hoc test. Kyn: kynurenic acid; RMP: resting membrane potential; SDR; spontaneous discharge rates; NLX: naloxone; LC: locus coeruleus; HZ: horizontal; OTH-cut: oblique to horizontal-cut; OTH: oblique to horizontal.
Fig 6.
Frequency and amplitude of sEPSCs in LC neurons of naïve (non-dependent) rats.
The samples traces of sEPSCs before and after application of naloxone (1 μM) in LC neurons of HZ (A), OTH-cut (B) and OTH (C) brain slices taken from naïve rats. Histograms indicate the mean frequency and amplitude of sEPSCs recorded before and after naloxone application. No significant alteration was observed in amplitude and frequency of sEPSCs in LC neurons of all slice forms following naloxone application. Data are expressed as mean ± SEM, n = 8 in each type of brain slice. Data were analyzed using paired Student’s t-test. NLX: naloxone; LC: locus coeruleus; sEPSCs: spontaneous excitatory post-synaptic currents; HZ: horizontal; OTH-cut: oblique to horizontal-cut; OTH: oblique to horizontal.
Fig 7.
Effect of naloxone on frequency and amplitude of EPSCs in LC neurons of morphine-dependent rats.
The samples traces of sEPSCs before and after application of naloxone (1 μM) in LC neurons of HZ (A), OTH-cut (B) and OTH (C) brain slices taken from morphine dependent rats. The slices were bathed in 5 μM morphine. There was no significant alteration in amplitude and frequency of sEPSCs in LC neurons of HZ and OTH-cut slices following naloxone application. As shown in histograms, the frequency of sEPSCs in LC neurons of OTH brain slices has significantly increased following naloxone application. However, no significant change was observed in the amplitude of sEPSCs after naloxone treatment in OTH brain slices. Histograms show the mean frequency and amplitude of sEPSCs recorded before and after naloxone application. Data are expressed as mean ± SEM, n = 8 in each type of brain slice, *** P < 0.001 versus before naloxone application. Data were analyzed using paired Student’s t-test. NLX: naloxone; LC: locus coeruleus; sEPSCs: spontaneous excitatory post-synaptic currents; HZ: horizontal; OTH-cut: oblique to horizontal-cut; OTH: oblique to horizontal.