Somatostatin Receptor 1 and 5 Double Knockout Mice Mimic Neurochemical Changes of Huntington's Disease Transgenic Mice

Selective degeneration of medium spiny neurons and preservation of medium sized aspiny interneurons in striatum has been implicated in excitotoxicity and pathophysiology of Huntington's disease (HD). However, the molecular mechanism for the selective sparing of medium sized aspiny neurons and vulnerability of projection neurons is still elusive. The pathological characteristic of HD is an extensive reduction of the striatal mass, affecting caudate putamen. Somatostatin (SST) positive neurons are selectively spared in HD and Quinolinic acid/N-methyl-D-aspartic acid induced excitotoxicity, mimic the model of HD. SST plays neuroprotective role in excitotoxicity and the biological effects of SST are mediated by five somatostatin receptor subtypes (SSTR1-5). and R6/2 mice. Conversely, the expression of somatostatin receptor subtypes, enkephalin and phosphatidylinositol 3-kinases were strain specific. SSTR1/5 appears to be important in regulating NMDARs, DARPP-32 and signaling molecules in similar fashion as seen in HD transgenic mice.This is the first comprehensive description of disease related changes upon ablation of G- protein coupled receptor gene. Our results indicate that SST and SSTRs might play an important role in regulation of neurodegeneration and targeting this pathway can provide a novel insight in understanding the pathophysiology of Huntington's disease

Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons.

The equilibrium potential for GABA-A receptor mediated currents (EGABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K+/Cl- co-transporter (KCC2) but a relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in EGABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on EGABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus EGABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in EGABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in EGABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in EGABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary

Sleep-deprivation induces changes in GABA(B) and mGlu receptor expression and has consequences for synaptic long-term depression.

Long term depression (LTD) in the CA1 region of the hippocampus, induced with a 20-Hz, 30 s tetanus to Schaffer collaterals, is enhanced in sleep-deprived (SD) rats. In the present study, we investigated the role of metabotropic glutamate receptors (mGluRs), γ-aminobutyric acid (GABA) B receptors (GABA(B)-Rs) and N-methyl-D-aspartic acid receptors (NMDARs) in the LTD of the population excitatory postsynaptic potential (pEPSP). The requirement of Ca(2+) from L- and T-type voltage-gated calcium channels (VGCCs) and intracellular stores was also studied. Results indicate that mGluRs, a release of Ca(2+) from intracellular stores and GABA(B)-Rs are required for LTD. Interestingly, while mGlu1Rs seem to be involved in both short-term depression and LTD, mGlu5Rs appear to participate mostly in LTD. CGP 55845, a GABA(B)-R antagonist, partially suppressed LTD in normally sleeping (NS) rats, while completely blocking LTD in SD rats. Moreover, GS-39783, a positive allosteric modulator for GABA(B)-R, suppressed the pEPSP in SD, but not NS rats. Since both mGluRs and GABA(B)-Rs seem to be involved in the LTD, especially in SD rats, we examined if the receptor expression pattern and/or dimerization changed, using immunohistochemical, co-localization and co-immunoprecipitation techniques. Sleep-deprivation induced an increase in the expression of GABA(B)-R1 and mGlu1αR in the CA1 region of the hippocampus. In addition, co-localization and heterodimerization between mGlu1αR/GABA(B)-R1 and mGlu1αR/GABA(B)-R2 is enhanced in SD rats. Taken together, our findings present a novel form of LTD sensitive to the activation of mGluRs and GABA(B)-Rs, and reveal, for the first time, that sleep-deprivation induces alterations in the expression and dimerization of these receptors

Western blot analysis of KCC2 in 3–5 day and 9–12 day old rat hippocampus.

<p>Total tissue lysate (20 μg) from CA1 region of hippocampus was subjected to immunoblot analysis using KCC2 specific antibodies. KCC2 expression was detected at the expected size of 140 kDa. Note the increased expression of KCC2 following TBS in the CA1 area of 9–12 day old rat hippocampus (panel B) while decreased expression of KCC2 in that of 3–5 day old rat hippocampus (panel A). KCC2 expression was not significantly changed in MCPG and MCPG plus TBS treated slices when compared to control in both 3–5 day old (n = 5) and 9–12 day old (n = 5) rat hippocampus. β -actin as loading control and densitometric analysis are shown in middle and bottom panel, respectively. Data are presented as mean ± SD, * P<0.05,***P<0.001.</p

MCPG blocks TBS-induced shifts in E_GABA in both juvenile and neonatal rat hippocampal neurons.

<p>MCPG (500 μM) was applied into superfusion medium 10 min prior to TBS conditioning. In panels A & B, the effects of MCPG on the TBS-induced shifts in E_GABA are shown from 9–12 and 3–5 day old rat hippocampal neurons, respectively. Data were obtained from two different CA1 neurons and holding potential for each individual cell was -60 mV. Note that MCPG blocked TBS-induced negative shift in E_GABA in both juvenile and neonatal rat hippocampal neurons. The whole-cell recording method was used.</p

Western blot analysis of NKCC1 in 3–5 day and 9–12 day old rat hippocampus.

<p>NKCC1 expression was seen at the expected size of 147 kDa. A) Western blot analysis showing the expression of NKCC1 in the CA1 region of hippocampus from control and treated slices from 3–5 day old rat (upper panel). Densitometric analysis reveals increase in NKCC1 expression in TBS treated, whereas, following MCPG treatment, NKCC1expression was comparable to that in control and no significant changes were seen in MCPG + TBS. B) Western blot analysis depicting comparative changes in the CA1 region of hippocampus in 9–12 day old rats, in control and following treatment (upper panel). Note the increased expression of NKCC1 upon TBS treatment whereas no discernable changes were seen upon MCPG and MCPG + TBS treatment when compared to control. β -actin as loading control and densitometric analysis are shown in middle and bottom panel, respectively. Data are presented as mean ± SD (n = 5 for each group), * P<0.05,** P<0.01.</p

Comparison of TBS-induced changes in E_GABA using whole-cell and perforated-patch recording methods.

<p>Traditional whole-cell patch clamping and perforated-patch recording were compared in two series of experiments. All the recordings were monitored for 10 min under 0.05 Hz stimulation before the TBS was delivered (at 0 min on X-axis). Note that shifts in E_GABA occur in hippocampal CA1 neurons of 9–12 day old rats following TBS in both methods (n = 6 for each). There was no statistical difference between records using the two methods. E_GABA was calculated using the method described in “Materials and Methods” section.</p