Rat Brain Slice Preparation Research Articles

Morphological and electrophysiological properties of lateral entorhinal cortex layers II and III principal neurons

The intrinsic electrophysiology and morphology of neurons from layers II and III of the lateral entorhinal cortex (EC) was investigated in a rat brain slice preparation by intracellular recording and biocytin labeling. Morphologically, we distinguished three groups of layer II principal neurons. The most numerous group included cells with multiple radiating dendrites that spread over layers II and I in a fan-like fashion. While morphologically "fan" neurons were similar to the "stellate" cells of the medial EC, electrophysiologically the fan cells lacked the persistent rhythmic subthreshold oscillations and the very pronounced time-dependent inward rectification typical of the stellate cells. The second group consisted of pyramidal cells that manifested regular spike firing and had a more negative resting potential and a longer spike duration than the fan cells. In the third group we included all those neurons that had diverse multipolar appearances distinct from the fan cells. Neurons in this group had electrophysiological profiles intermediate between those of the fan and pyramidal cells. All neurons recorded in layer III were pyramidal in shape with a basal dendritic tree that could extend into layer V and an axon that could also give off collaterals into layer V. Electrophysiologically, layer III pyramidal cells were very similar to those of layer II. On the basis of these and other data we suggest that in different EC regions layer II neurons may be conducting more input-dependent specialized processing, while cells from layer III may perform a more global or generalized function.

Effects of orexins/hypocretins on neuronal activity in the paraventricular nucleus of the thalamus in rats in vitro

Orexin-A (ORX-A) and orexin-B (ORX-B), also called hypocretin-1 and hypocretin-2, respectively, act upon orexin 1 (OX 1R) and orexin 2 (OX 2R) receptors, and are involved in the regulation of sleep-wakefulness and energy homeostasis. Orexin neurons in the lateral hypothalamic perifornical region project heavily to the paraventricular nucleus of the thalamus (PVT), which is deeply involved in the control of motivated behaviors. In the present study, electrophysiological and cytosolic Ca 2+ concentration ([Ca 2+] i ) imaging studies on the effects of ORX-A and ORX-B on neurons in the PVT were carried out in rat brain slice preparations. ORX-A and/or ORX-B were applied extracellularly in the perfusate. Extracellular recordings showed that about 80% of the PVT neurons were excited dose-dependently by both ORX-A and ORX-B at concentrations of 10 −8 to 10 −6 M, and the increase in firing rate was about three times larger for ORX-B than for ORX-A at 10 −7 M. When both ORX-A and ORX-B were applied simultaneously at 10 −7 M, the increase in firing rate was almost equal to that of ORX-B at 10 −7 M, suggesting that the PVT neurons do not show a high affinity to ORX-A which is expected if they have OX 1R receptors. The excitatory effect of ORX-B was seen in low Ca 2+ and high Mg 2+ ACSF as well as in normal ACSF, and the increase in firing rate was greater in low Ca 2+ and high Mg 2+ ACSF than in normal ACSF. [Ca 2+] i imaging studies demonstrated that [Ca 2+] i was increased in about 50% of the PVT neurons by both 10 −7 M ORX-A and ORX-B with a stronger effect for ORX-B, and the increase in [Ca 2+] i induced by ORX-B was abolished in Ca 2+-free ACSF, suggesting that ORX-B does not release Ca 2+ from intracellular Ca 2+ stores. Subsequent whole cell patch clamp recordings revealed that an after hyperpolarization seen following each action potential in normal ACSF disappeared in Ca 2+-free ACSF, and the mean magnitude of the depolarization induced by ORX-B was same in normal, Ca 2+-free and TTX-containing Ca 2+-free ACSFs. Furthermore, ORX-B-induced depolarization was reversed to hyperpolarization when membrane potential was lowered to about −97 mV, and an increase of extracellular K + concentration from 4.25 to 13.25 mM abolished the ORX-B-induced depolarization, indicating that the ORX-B-induced depolarization is associated with an increase in the membrane resistance resulting from a closure of K + channels. These results suggest that orexins depolarize and excite post-synaptically PVT neurons via OX 2R receptors, and that orexin-activated PVT neurons play a role in the integration of sleep-wakefulness and energy homeostasis, and in the control of motivated behaviors.

Prolactin releasing peptides modulate background firing rate and milk-ejection related burst of oxytocin cells in the supraoptic nucleus

The hypothalamic dorsomedial nucleus is suggested to be a final relay site for the afferent pathway of milk-ejection reflex. Existence of prolactin releasing peptide-immunoreactive cells in the dorsomedial nucleus and synaptic contact of prolactin releasing peptide-immunoreactive terminals with oxytocin cells was reported. Experiments were done to test the effect of prolactin releasing peptide on the electrical activity of oxytocin cells in the supraoptic nucleus. In rat brain slice preparations, oxytocin cells were unresponsive to the peptide. In lactating rats, although lateral ventricular injection of prolactin releasing peptide (20 nmol) was ineffective, a hundred nanomoles of the peptide increased basal activity and amplitude of milk-ejection related burst firing of oxytocin cells. Cells responded to lateral ventricular injection of peptides were unresponsive to direct application of peptides by pressure ejection from the recording electrode. These results suggest that prolactin releasing peptide may modulate electrical activity of oxytocin cells not through its direct action on oxytocin cells but through its action on area other than supraoptic nucleus.

Activation of β1-Adrenoceptors Excites Striatal Cholinergic Interneurons through a cAMP-Dependent, Protein Kinase-Independent Pathway

The role of noradrenergic neurotransmission was analyzed in striatal cholinergic interneurons. Conventional intracellular and whole-cell patch-clamp recordings were made of cholinergic interneurons in rat brain slice preparations. Bath-applied noradrenaline (NA) (1-300 microm) dose-dependently induced both an increase in the spontaneous firing activity and a membrane depolarization of the recorded cells. In voltage-clamped neurons, an inward current was induced by NA. This effect was not prevented by alpha-adrenoceptor antagonists, whereas it was mimicked by the beta-adrenoceptor agonist isoproterenol and blocked by the beta1 antagonists propranolol and betaxolol. Interestingly, forskolin, activator of adenylate cyclase, mimicked and occluded the membrane depolarization obtained at saturating doses of both dopamine and NA. Accordingly, SQ22,536, a selective adenylate cyclase inhibitor, reduced the response to NA. Analysis of the reversal potential of the NA-induced current did not provide homogeneous results, indicating the involvement of multiple membrane conductances. Because cAMP is known to modulate Ih, the effects of ZD7288, a selective inhibitor of Ih current, were examined on the NA-induced membrane depolarization/inward current. ZD7288 mostly reduced the response to NA. However, both KT-5720 and H-89, selective protein kinase A (PKA) blockers, failed to prevent the excitatory action of NA. Likewise, calphostin C, antagonist of PKC, genistein, inhibitor of tyrosine kinase, and 8-Bromo-cGMP, blocker of PKG, did not affect the response to NA. Finally, double-labeling experiments combining beta1-adrenoceptor and choline acetyltransferase immunocytochemistry by means of confocal microscopy revealed a strong beta1-adrenoceptor labeling on cholinergic interneurons. We conclude that NA depolarizes striatal cholinergic interneurons via beta1-adrenoceptor activation, through a cAMP-dependent but PKA-independent mechanism.

Intrinsic Role of Polysialylated Neural Cell Adhesion Molecule in Photic Phase Resetting of the Mammalian Circadian Clock

The suprachiasmatic nuclei (SCN), the location of the mammalian circadian clock, are one of the few adult brain regions that express the highly polysialylated form of neural cell adhesion molecule (PSA-NCAM). A role for the polysialic acid (PSA) component of PSA-NCAM, which is known to promote tissue plasticity, has been reported for photic entrainment of circadian rhythmicity in vivo. The in vivo results, however, do not discriminate between PSA acting upstream or downstream of the glutamatergic synapses that convey photic information to the SCN. To address this key issue, we exploited an in vitro rat brain slice preparation that retains robust circadian function. As in the intact SCN, PSA levels in the isolated SCN are rhythmic, with higher levels during the early subjective day and lower levels during subjective night. Importantly, bath application of glutamate to SCN slices rapidly and transiently increases PSA levels during both the subjective day and night. Pretreating the slices with endoneuraminidase, which selectively removes PSA from NCAM and thereby prevents this increase, abolishes glutamate- and optic chiasm stimulation-induced phase delays of the SCN circadian neuronal activity rhythm. These results support the hypothesis that PSA expression in the SCN is controlled by both the circadian clock and photic input to the clock and that expression of PSA in the SCN is critical for photic-like phase shifts of the clock. Together, these results establish that such actions of PSA are manifested downstream from presynaptic retinohypothalamic terminals and therefore are intrinsic to the SCN itself.

Activation of group III metabotropic glutamate receptors presynaptically reduces both GABAergic and glutamatergic transmission in the rat globus pallidus

To investigate the role of group III metabotropic glutamate receptors (mGluRs) in the globus pallidus (GP), whole-cell recordings were performed using rat brain slice preparations. Application of the group III mGluRs specific agonist l(+)-2-amino-4-phosphonobutyric acid ( l-AP4) suppressed the amplitude of striatal stimulation-induced IPSCs and internal capsule stimulation-induced EPSCs in most of the GP neurons that were capable of generating repetitive firing without spike accommodation. The suppression of IPSCs and EPSCs was accompanied by an increase in the paired-pulse ratio. The l-AP4 effects were antagonized by ( R, S)-alpha-cyclopropyl-4-phosphophenylglycine, a blocker for group II/III mGluRs. l-AP4 reduced the frequency of mIPSCs and mEPSCs without changing their amplitude distribution. l-AP4 failed to change iontophoretic glutamate induced responses. These results suggest that the subthalamo-pallidal glutamatergic input might homo- and hetero-synaptically control GABAergic and glutamatergic transmission in the GP.

Presynaptic inhibition of synaptic transmission by adenosine in rat subthalamic nucleus in vitro

Whole-cell patch clamp recordings were made from the subthalamic nucleus in rat brain slice preparations to examine the effect of adenosine on inhibitory and excitatory synaptic transmission. Adenosine reversibly inhibited both GABA-mediated inhibitory and glutamate-mediated excitatory postsynaptic currents. Adenosine at 100 μM reduced the amplitude of inhibitory and excitatory postsynaptic currents by 42±5% and 34±6%, respectively. Reductions in the amplitude of both inhibitory and excitatory postsynaptic currents were accompanied by increases in paired-pulse ratios. In addition, adenosine decreased the frequency of spontaneous miniature excitatory postsynaptic currents but had no effect on their amplitude. These results are consistent with a presynaptic site of action. The adenosine A 1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine completely reversed the adenosine-induced attenuation of inhibitory and excitatory postsynaptic currents, but 8-cyclopentyl-1,3-dipropylxanthine alone had no effect on synaptic currents evoked at 0.1 Hz. However, 8-cyclopentyl-1,3-dipropylxanthine inhibited a time-dependent depression of excitatory postsynaptic currents that was normally observed in response to a 5 Hz train of stimuli, suggesting that endogenous adenosine could be released during higher frequencies of stimulation. These results suggest that adenosine inhibits synaptic release of GABA and glutamate by stimulation of presynaptic A 1 receptors in the subthalamic nucleus.

Mitochondrial Complex Inhibitors Preferentially Damage Substantia Nigra Dopamine Neurons in Rat Brain Slices

Using a rat brain slice preparation, we investigated the role of energy impairment on the selective loss of dopamine neurons in the substantia nigra (SN). Brain slices (400 μm) were incubated at 35°C for 2 h in the presence or absence of mitochondrial complex inhibitors, rotenone, MPP +, 3-nitropropionic acid, and antimycin A. Slices were also incubated in rotenone with excitatory amino acid (EAA) receptor antagonists, MK-801 and CNQX, to determine whether rotenone-induced damage was mediated by EAAs. The slices were then fixed, recut into 30-μm sections, and immunolabeled for tyrosine hydroxylase (TH) to identify catecholamine neurons and to quantify loss of TH-labeled dendrites after treatment. Quantitative comparison was made between SN dopamine neurons, in which rotenone-induced dendrite loss was severe, and hypothalamic A11 dopamine neurons, which were spared. Adjacent sections that were immunolabeled for calbindin or stained with cresyl violet also revealed a striking dendritic degeneration of SN neurons in rotenone-exposed slices, whereas noncatecholamine neurons, such as those in the perifornical nucleus (PeF), were more resistant. Preferential damage to SN dopamine neurons was also evident with other mitochondrial complex inhibitors, MPP + and antimycin A. EAA receptor antagonists provided partial protection to SN neurons in slices incubated with rotenone (3 μM). The particular vulnerability of SN dopamine neurons in the slice is consistent with the vulnerability of SN in Parkinson's disease. The selective effect of mitochondrial complex inhibition in SN dopamine neurons implies a fundamental deficit in the capacity of these neurons to defend against toxic insult.

Mesencephalic trigeminal neurons are innervated by nitric oxide synthase-containing fibers and respond to nitric oxide

In the present study we found that mesencephalic trigeminal (Mes-V) neurons of the rat are innervated by nitrergic fibers and that nitric oxide (NO) modifies the electrophysiological properties of these cells. Mes-V neurons were surrounded by a network of fibers that contained neuronal nitric oxide synthase (nNOS); these fibers gave rise to terminal-, bouton-like structures which ended in Mes-V cells bodies. These cells, which did not display nNOS-like immunoreactivity were immunoreactive to a cGMP antibody. By performing intracellular recordings in the adult rat brain slice preparation, the effects of diethylenetriamine/NO adduct (DETA/NO) applications were examined. DETA/NO induced a depolarization that averaged 2.2 mV (range: 1–6 mV) in nine of 22 neurons. In 15 of 22 neurons (68% of the cells), there was a decrease in current threshold from 0.74 to 0.60 nA (19%; P<0.001). The excitatory effects of DETA/NO were abolished by ODQ, a blocker of soluble guanylate cyclase. Input resistance ( R in) decreased in 80% of the cells from a mean of 24.8 to 20.6 MΩ (17%; P<0.001) and the membrane time constant ( τ m) decreased from 7.5 to 5.6 ms (25%; P<0.05). The ‘sag’ seen in the membrane response of these cells to current pulses was augmented during DETA/NO application. These findings indicate that there is a nitrergic innervation of Mes-V neurons and that these sensory cells are target for NO that may act on them as an excitatory neuromodulator promoting the synthesis of intracellular cGMP.

Effects of dynorphin on rat entopeduncular nucleus neurons in vitro

The entopeduncular nucleus (EP) receives dense neostriatal afferent axons that contain dynorphin (DYN, an endogenous κ-receptor agonist), in addition to GABA and substance P. To examine the role of DYN in the EP, whole-cell recordings were performed in rat brain slice preparations. Based on the physiological and morphological characteristics, all the neurons recorded were similar to the Type-I EP neuron described in a previous study. The κ-receptor agonist dynorphin A (1-13) (DYN13) hyperpolarized and decreased the input resistance of approximately one-quarter of the EP neurons examined. The hyperpolarization was due to an increase in potassium conductance since current–voltage relationship curves obtained before and after DYN13 application crossed at the potassium equilibrium potential. In the presence of the glutamate blocker 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide and 3-(2-carboxypiperzin-4-yl)-propyl-1-phosphonic acid in artificial cerebrospinal fluid, stimulation of the globus pallidus evoked bicuculline-sensitive multi-component GABAergic responses in EP neurons. Application of DYN13 equally reduced the amplitudes of the short-latency response, conceivably evoked by pallido-EP axons, and the medium-latency response, conceivably evoked by striato-EP axons. These effects were reversed by bath application of a non-selective opioid antagonist naloxone or by a κ-opioid receptor-selective antagonist nor-binaltorphimine dihydrochloride (nor-BNI), but not by the ∂-antagonist naltrindole or the μ-antagonist D-Phe-Cys-Tyr- D-Trp-Orn-Thr-Pen-Thr-NH 2. DYN13 also reduced the frequency of tetrodotoxin-insensitive miniature-inhibitory postsynaptic potential (mIPSPs) without changing their amplitude distributions. The decrease of the frequency of mIPSPs was reversible upon washing and was also completely blocked by nor-BNI. The results of the present study on the EP indicated that DYN released from striatal axons might exert at least three different effects on these target nuclei. Firstly, DYN might provide negative feedback regulation of striatal GABAergic outputs at their termination sites. Secondly, DYN released from the striatal terminals might diffuse to the pallidal terminals, regulating their GABA release. Thirdly, DYN might exert a direct inhibition of EP neurons. Thus, DYN released from striatal axons might control the activity of EP neurons by reducing the GABAergic transmission and also by hyperpolarizing postsynaptic membrane.

Effects of paired-pulse and repetitive stimulation on neurons in the rat medial geniculate body

Many behaviorally relevant sounds, including language, are composed of brief, rapid, repetitive acoustic features. Recent studies suggest that abnormalities in producing and understanding spoken language are correlated with abnormal neural responsiveness to such auditory stimuli at higher auditory levels [Tallal et al., Science 271 (1996) 81–84; Wright et al., Nature 387 (1997) 176–178; Nagarajan et al., Proc. Natl. Acad. Sci. USA 96 (1999) 6483–6488] and with abnormal anatomical features in the auditory thalamus [Galaburda et al., Proc. Natl. Acad. Sci. USA 91 (1994) 8010–8013]. To begin to understand potential mechanisms for normal and abnormal transfer of sensory information to the cortex, we recorded the intracellular responses of medial geniculate body thalamocortical neurons in a rat brain slice preparation. Inferior colliculus or corticothalamic axons were excited by pairs or trains of electrical stimuli. Neurons receiving only excitatory collicular input had tufted dendritic morphology and displayed strong paired-pulse depression of their large, short-latency excitatory postsynaptic potentials. In contrast, geniculate neurons receiving excitatory and inhibitory collicular inputs could have stellate or tufted morphology and displayed much weaker depression or even paired-pulse facilitation of their smaller, longer-latency excitatory postsynaptic potentials. Depression was not blocked by ionotropic glutamate, GABA A or GABA B receptor antagonists. Facilitation was unaffected by GABA A receptor antagonists but was diminished by N-methyl- D-aspartate (NMDA) receptor blockade. Similar stimulation of the corticothalamic input always elicited paired-pulse facilitation. The NMDA-independent facilitation of the second cortical excitatory postsynaptic potential lasted longer and was more pronounced than that seen for the excitatory collicular inputs. Paired-pulse stimulation of isolated collicular inhibitory postsynaptic potentials generated little change in the second GABA A potential amplitude measured from the resting potential, but the GABA B amplitude was sensitive to the interstimulus interval. Train stimuli applied to collicular or cortical inputs generated intra-train responses that were often predicted by their paired-pulse behavior. Long-lasting responses following train stimulation of the collicular inputs were uncommon. In contrast, corticothalamic inputs often generated long-lasting depolarizing responses that were dependent on activation of a metabotropic glutamate receptor. Our results demonstrate that during repetitive afferent firing there are input-specific mechanisms controlling synaptic strength and membrane potential over short and long time scales. Furthermore, they suggest that there may be two classes of excitatory collicular input to medial geniculate neurons and a single class of small-terminal corticothalamic inputs, each of which has distinct features.

Abused Inhalants and Central Reward Pathways

Inhalant abuse remains a significant health problem among the younger segment of society. In fact, the use of inhalants in this population trails only that of nicotine, alcohol, and marijuana. Toluene is a common ingredient in many of the substances sought out for inhalation abuse, apparently for its euphorigenic and hallucinogenic effects. Because drugs of abuse share the common property of altering the activity of mesolimbic dopamine neurons, it is reasonable to suspect that toluene-induced changes in this CNS pathway may underlie its abuse potential. Here we will provide in vivo and in vitro electrophysiological data and behavioral evidence linking toluene exposure in rats to activation of mesolimbic dopamine neurons. Exposure of rats to 11,000 ppm of inhaled toluene produced time-dependent activation of dopamine neurons within the midbrain ventral tegmental area (VTA). In the rat brain slice preparation, perfusion with toluene (23-822 microM) also evoked an increase in activity of both dopamine and nondopamine neurons within the VTA. These excitatory effects could not be found in adjacent non-VTA nuclei, nor were they sensitive to the glutamate antagonists CGS19755 or CNQX. In behavioral studies, systemic administration of toluene produced a dose-dependent locomotor hyperactivity that was attenuated by either pretreatment with the D2 dopamine receptor antagonist remoxipride or by 6-hydroxydopamine lesions of the nucleus accumbens. These findings show that toluene can activate dopamine neurons within the mesolimbic reward pathway, an effect that may underlie the abuse potential of inhaled substances containing toluene.

Pharmacological identification of inward current evoked by dopamine in rat subthalamic neurons in vitro

Dopaminergic mechanisms in the subthalamic nucleus (STN) are implicated in the pathophysiology of Parkinson’s disease. Here, electrophysiological responses of STN neurons to dopamine (DA) were investigated by using whole-cell patch-clamp recordings in the rat brain slice preparation. Under current-clamp, DA depolarized membrane potential and increased the frequency of spontaneous action potentials of STN neurons. Under voltage-clamp, DA (3–300 μM) produced a reversible concentration-dependent inward current ( I DA; 6–40 pA) with an EC 50 of 13 μM. This DA-induced current had a negative slope conductance which reversed at −102 mV. It was partially reduced by barium and by superfusion with an elevated concentration of extracellular K +. Moreover, TTX and glutamate receptor antagonists (CNQX and AP5) did not significantly affect the DA responses, indicating that I DA is not dependent upon afferent synaptic activity in the STN. Quinpirole, a D 2 receptor agonist, mimicked the DA action more effectively than did the D 1 agonist SKF-38393. The D 2 antagonist sulpiride, but not the D 1 antagonist SCH-23390, blocked responses induced by DA. Intracellular application of G-protein inhibitor GDP-β-S also suppressed I DA. GTP-γ-S, added to the pipette solution, evoked a sustained inward shift in the absence of DA. These results suggest that DA increases the activity of STN neurons via activation of G-protein-coupled D 2-like receptors which reduce a K + conductance.

Properties of the pathways from the lateral amygdal nucleus to basolateral nucleus and amygdalostriatal transition area.

Studies have revealed that the amygdala formation is involved in emotional learning, attention, and autonomic functions. Although intra-amygdala connections have been described anatomically, the functional characteristics of these connections are not well understood. We used a rat brain slice preparation with a voltage-sensitive imaging system to compare the electrophysiological characteristics of intra-amygdala pathways. Electrical stimuli delivered to the lateral nucleus (La) caused the optical signal to propagate to basolateral nucleus (BL) and amygdalostriatal transition area (AStr), but not the central nucleus (Ce), consistent with previous anatomical studies, including the recently characterized projections from La to AStr. The velocity of propagation of the evoked potential along the La-AStr pathway was significantly faster than that along the La-BL pathway. In addition, the efficiency of the signal transmission (determined by the rate of decay) along the La-AStr pathway was higher than that along the La-BL pathway. Also, AStr possessed a distinct property of temporal summation of La signals. On the other hand, the La-BL pathway possessed a significantly higher sensitivity to bicuculline/picrotoxin and a stronger paired-pulse inhibition than the La-AStr pathway. Furthermore, the La-BL pathway expressed a higher D-2-amino-5-phosphonovaleric acid (a NMDA blocker) sensitivity than the La-AStr pathway. These results suggest that the La-AStr pathway, which conducts signals with high velocity and less attenuation, may be involved in rapid reflexive responses during fear-induced behavior, whereas the La-BL pathway facilitates signal integration and learning.

Leptin affects the electrical activity of neurones in the hypothalamic supraoptic nucleus

The present experiments were undertaken to examine whether leptin affects the electrical activity of neurones in the supraoptic nucleus (SON) by using brain slice preparation of male Wistar and obese Zucker rats. Bath application of leptin (10−8 − 10−12 M) induced mainly inhibitory response in SON neurones of Wistar rats, although a minority showed excitation. These effects were observed in both continuously and phasically active cells. The inhibitory effect of leptin still persisted in low Ca2+, high Mg2+ medium. Bath application of tolbutamide, which is known to inhibit ATP-sensitive potassium channel activity, did not reverse the inhibitory effect of leptin on SON neurones. The effect of bath application of leptin was also tested in SON neurones of obese Zucker rats. Although leptin still affected the electrical activity of some SON neurones of Zucker rats, the proportion of unaffected neurones was significantly higher than in Wistar rats. The results suggest that leptin may inhibit the secretion of both oxytocin and vasopressin by inhibiting the electrical activity of neurones in the SON via direct action. This inhibitory effect of leptin may be exerted through mechanisms other than activation of ATP-sensitive potassium channels.

Catecholamine neuron groups in rat brain slices differ in their susceptibility to excitatory amino acid induced dendritic degeneration.

We investigated whether specific types of catecholamine neurons were differentially vulnerable to damage induced by excitatory amino acids (EAAs) in vitro in a rat brain slice preparation. Brain slices, 300 micro m thick, were cut horizontally, exposed to either N-methyl-D-aspartate (NMDA) or kainic acid (KA) for 2h, fixed and then cut into thin (30 micro m) sections in the same (horizontal) plane as the slice. The sections were immunolabelled for tyrosine hydroxylase to identify different groups of catecholamine neurons (substantia nigra (SN), paranigral (PN), interfascicular (IF) and hypothalamic A11, A13 and A14) which exhibited prominent dendritic projections in the horizontal plane. Loss of dendrites was used as a sensitive index of damage that precedes the loss of the cell body. Catecholamine neurons differed strikingly in their vulnerability of EAA-induced dendrite degeneration. The most vulnerable were those in the dorsal tier of the SN, whereas the most resistant were those in the hypothalamic A11 group. For example, in the dorsal tier of SN, NMDA (50 micro M) reduced the proportion of neurons with dendrites from 64% (+/- 8% SEM) in controls to 13% (+/- 7%) whereas the majority of A11 neurons (69 +/- 10%) retained their dendrites compared to controls (89% +/- 8%). The other groups of catecholamine neurons exhibited intermediate vulnerability. An essentially similar pattern of differential vulnerability was observed with KA. An understanding of the cellular mechanisms that underlie the particular vulnerability of SN neurons in the slice will aid the discovery of pharmacological therapies to prevent or slow the pathological process in neurodegenerative diseases which involve these neurons.

Neostriatal and globus pallidus stimulation induced inhibitory postsynaptic potentials in entopeduncular neurons in rat brain slice preparations

Recent anatomical studies revealed that the entopeduncular nucleus of the rat receives GABAergic inputs from both the neostriatum and the globus pallidus. The present study was undertaken to reveal the physiological features of these inputs using the intracellular recording method in rat brain slice preparations. Most of the entopeduncular nucleus neurons generated repetitive firing without spike accommodation with intracellular current stimulation and thus were classified as Type-I. A small number of neurons were classified as Type-II since they generated spikes with pronounced accommodation. Most of the Type-I, but none of Type-II, entopeduncular nucleus neurons exhibited monosynaptic GABAergic inhibitory postsynaptic potentials (IPSPs) after stimulation of the neostriatum and the globus pallidus. Neostriatal stimulation induced long latency IPSPs while pallidal stimulation induced long latency IPSPs compounded with short latency IPSPs. The IPSPs were mediated by GABA A receptors. The unitary IPSPs to striatal stimulation were small while those to pallidal stimulation were large in amplitude and able to reset ongoing rhythmic firing. The short latency IPSPs induced by pallidal stimulation reversed at a somatic membrane potential that was a few millivolts more depolarized than the long latency IPSPs, suggesting that the striatal inputs were evoked in more distal portions of the neurons than the pallidal inputs. Repetitive activation of these inputs resulted in a poor amplitude summation but a prolongation of the duration of the IPSPs. The results of the present study indicate that the pallidal projection to the entopeduncular nucleus is physiologically significant and that the neostriatum and the globus pallidus play important roles in controlling the activity of the entopeduncular nucleus, although in different ways.

Differential short-term changes in GABAergic or glycinergic synaptic efficacy on rat hypoglossal motoneurons.

Using whole cell patch-clamp recording from hypoglossal motoneurons of a neonatal rat brain slice preparation, we investigated short-term changes in synaptic transmission mediated by GABA or glycine. In 1.5 mM extracellular Ca(2+) [Ca(2+)](o), pharmacologically isolated GABAergic or glycinergic currents were elicited by electrical stimulation of the reticular formation. At low stimulation frequency, glycinergic currents were larger and faster than GABAergic ones. GABAergic currents were strongly facilitated by pulse trains at 5 or 10 Hz without apparent depression. This phenomenon persisted after pharmacological block of GABA(B) receptors. Glycinergic currents were comparatively much less enhanced than GABAergic currents. One possible mechanism to account for this difference is that GABAergic currents decayed so slowly that consecutive responses summated over an incrementing baseline. However, while synaptic summation appeared at > or =10-Hz stimulation, at 5 Hz strong facilitation developed with minimal summation of GABA-mediated currents. Glycinergic currents decayed so fast that summation was minimal. As [Ca(2+)](o) is known to shape short-term synaptic changes, we examined if varying [Ca(2+)](o) could differentially affect facilitation of GABA- or glycine-operated synapses. With 5 mM [Ca(2+)](o), the frequency of spontaneous GABAergic or glycinergic currents appeared much higher but GABAergic current facilitation was blocked (and replaced by depression), whereas glycinergic currents remained slightly facilitated. [Ca(2+)](o) manipulation thus brought about distinct processes responsible for facilitation of GABAergic or glycinergic transmission. Our data therefore demonstrate an unexpectedly robust, short-term increase in the efficiency of GABAergic synapses that can become at least as effective as glycinergic synapses.

Thalamocortical connectivity in a rat brain slice preparation: participation of the ventrobasal complex to synchronous activities

We studied the synchronous cortical and thalamic activities induced by low (0.5–1 mM) and high (50–100 mM) concentrations of the K+ channel blocker 4-aminopyridine (4AP) in a rat thalamocortical preparation. The presence of reciprocal thalamocortical connectivity was documented by diffusion of the fluorescent tracer Di-IC18 between the somatosensory cortex and the ventrobasal complex (VB) of the thalamus in vitro. Functional reciprocal connectivity was also demonstrated by stimulating the cortical middle-deep layers (which elicited orthodromic responses in VB) or the VB (which induced orthodromic and antidromic responses in the cortex). Spontaneous field potentials were not recorded in either the thalamus or cortex in control medium. Low concentrations of 4AP produced local spindle-like rhythmic oscillations in cortex and VB (duration = 0.4–3.5 s; frequency = 9–16 Hz). In contrast, high concentrations of 4AP induced widespread ictal-like epileptiform discharges (duration = 8–45 s) characterised by lsquo ‘tonic’ component followed by a period of ‘clonic’ discharges in both cortex and VB. Spindle-like activity was abolished in cortex and thalamus by applying the excitatory amino acid receptor antagonist kynurenic acid in VB. In contrast, the same procedure exacerbated ictal-like discharges, while depressing VB activity. Our results indicate that thalamus and cortex can produce synchronous activities in this in vitro thalamocortical network: spindle-like rhythmic oscillations are generated at the thalamic level and imposed upon the cortical network whereas ictal-like discharges have a cortical origin and are modulated by the thalamic network activity. In addition, we have shown that it is possible to preserve reciprocal projections between cortex and thalamus in an in vitro rat slice preparation that could be a valuable tool to study epileptic-prone rat strains.

Thalamocortical connectivity in a rat brain slice preparation: participation of the ventrobasal complex to synchronous activities

We studied the synchronous cortical and thalamic activities induced by low (0.5–1 mM) and high (50–100 mM) concentrations of the K + channel blocker 4-aminopyridine (4AP) in a rat thalamocortical preparation. The presence of reciprocal thalamocortical connectivity was documented by diffusion of the fluorescent tracer Di-IC 18 between the somatosensory cortex and the ventrobasal complex (VB) of the thalamus in vitro. Functional reciprocal connectivity was also demonstrated by stimulating the cortical middle-deep layers (which elicited orthodromic responses in VB) or the VB (which induced orthodromic and antidromic responses in the cortex). Spontaneous field potentials were not recorded in either the thalamus or cortex in control medium. Low concentrations of 4AP produced local spindle-like rhythmic oscillations in cortex and VB (duration=0.4–3.5 s; frequency=9–16 Hz). In contrast, high concentrations of 4AP induced widespread ictal-like epileptiform discharges (duration=8–45 s) characterised by a ‘tonic’ component followed by a period of ‘clonic’ discharges in both cortex and VB. Spindle-like activity was abolished in cortex and thalamus by applying the excitatory amino acid receptor antagonist kynurenic acid in VB. In contrast, the same procedure exacerbated ictal-like discharges, while depressing VB activity. Our results indicate that thalamus and cortex can produce synchronous activities in this in vitro thalamocortical network: spindle-like rhythmic oscillations are generated at the thalamic level and imposed upon the cortical network whereas ictal-like discharges have a cortical origin and are modulated by the thalamic network activity. In addition, we have shown that it is possible to preserve reciprocal projections between cortex and thalamus in an in vitro rat slice preparation that could be a valuable tool to study epileptic-prone rat strains.