Voltage-dependent Kinetics of N-Methyl-d-aspartate Synaptic Currents in Rat Cerebellar Granule Cells

Decay kinetics of N -methyl-d-aspartate excitatory postsynaptic currents (NMDA-EPSCs) have been voltage-dependent in some, but not all neurons studied so far, and almost no information has been available on the voltage-dependence of the rising phase. In this work we investigated the effect of membrane potential on rising and decay kinetics of the NMDA-EPSC in cerebellar granule cells using the tight-seal whole-cell recording technique. NMDA-EPSCs were evoked by electrical mossy fibre stimulation in the presence of 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione, 1.2 mM Mg²⁺ and 5 μM glycine. The rate of rise of NMDA-EPSCs remained substantially unchanged when the cell was depolarized, indicating that the limiting step of channel opening was voltage-insensitive. The NMDA-EPSC, however, flattened around the peak and the time-to-peak increased. This observation was explained by the influence of decay. Decay was biphasic and slowed down with membrane depolarization. Moreover, the fast component of decay increased less than the slow component. This complex voltage-dependence may extend the integrative role of the NMDA current during synaptic transmission.     

Differential Long-lasting Potentiation of the NMDA and Non-NMDA Synaptic Currents Induced by Metabotropic and NMDA Receptor Coactivation in Cerebellar Granule Cells

Whole-cell patch-clamp recordings in rat cerebellar slices were used to investigate the effect of metabotropic glutamate receptor activation on mossy fibre-granule cell synaptic transmission. Transient application of 20 μM 1 S , 3 R -1 -aminocyclopentane-1,3-dicarboxylic acid simultaneously with low-frequency NMDA receptor activation induced long-lasting non-decremental potentiation of both NMDA and non-NMDA receptor-mediated synaptic transmission. Potentiation could be prevented by application of the metabotropic glutamate receptor antagonist (+)- O -methyl-4-carboxyphenyl-glycine at 500 μM. Characteristically, NMDA potentiation was two to three times as large as non-NMDA current potentiation, occurred only in a slow subcomponent, and was voltage independent. This result demonstrates a pivotal role of NMDA receptors in the metabotropic potentiation of transmission, which may be important in regulating cerebellar information processing.     

Sodium and Calcium Currents in Glial Cells of the Mouse Corpus Callosum Slice

We studied Na⁺ and Ca²⁺ currents in glial cells during the development of the corpus callosum in situ. Glioblasts and oligodendrocytes from frontal brain slices of postnatal day (P) 3 to P18 mice were identified based on morphological and ultrastructural features after characterization of the currents with the patch-clamp technique. Slices from P3-P8 mice contained predominantly glioblasts with immature morphological features. These cells showed Na⁺ and Ca²⁺ currents, but the population with these currents decreased between P3 and P8. Na⁺ currents were blocked in Na⁺-free bathing solution and in the presence of tetrodotoxin, Ca²⁺ currents were only observed when a high concentration of extracellular Ba²⁺ was present. The cells from the corpus callosum of P10 – P18 mice predominantly had morphological features of oligodendrocytes. In these cells, which in some cases were shown to form myelin, neither Na⁺ nor Ca²⁺ currents were detected. To compare these in situ results with those from the electrophysiologically and immunocytochemically well-characterized cultured glial cells, we determined the expression pattern of stage-specific antigens in the corpus callosum in situ. The first O4 antigen-positive glial precursors were observed at P1, the earliest stage examined. The oligodendrocytic antigens O7 and O10 appeared at P6 and P14, respectively, and prominent labelling with the corresponding markers was seen at P12 and P18, respectively. Despite the existence of numerous mature, O10-positive oligodendrocytes at P18, which expressed Ca²⁺ channels in vitro , we failed to detect Ca²⁺ currents in situ at this stage.     

Single Channel Recording from Glial Cells on the Untreated Surface of the Frog Optic Nerve

The patch clamp technique has been used to record single channel currents from the untreated surface of the intact frog optic nerve after the meninges and basal lamina have been mechanically removed. Cells filled via dialysis with Lucifer yellow (LY) from the patch pipette had a typical astrocyte morphology and were dye-coupled to adjacent astrocytes. This is consistent with the electron-microscopic observation that all the cells on the surface of this nerve are astrocytes. Two types of ion channels were studied in detached patches. One, identified as a K+ channel, had a conductance of 88 +/- 4 (S.E.) n=9 pS and an equilibrium potential of -59 +/- 8 mV in physiological K+ solutions. The steady-state open probability was not significantly altered by changing the membrane potential. A second channel had a large conductance of 300 - 1200 pS, a reversal potential of approximately 0 mV in symmetrical and non-symmetrical solutions, and was open only in the voltage range of +/-20 mV. These are the characteristics of a large anionic channel described in other preparations including cultured astrocytes.     

Voltage-gated Currents in Rabbit Retinal Astrocytes

The voltage-gated currents of the astrocytes associated with the retinal capillaries of the rabbit retina were studied using whole-cell patch clamp recording. The resting potential of these cells was −70 ± 4.8 mV (mean ± SEM; n = 54), and the input resistance and cell capacitance were 558 ± 3.6 MΩ and 19.5 ± 1.8 pF respectively. Depolarization to potentials positive to −50 mV evoked rapidly activating inward and outward currents. The inward current was transient, eliminated by substitution of choline for Na⁺ in the bathing solution, and reduced by 50% in the presence of 1 μM tetrodotoxin. The time-to-peak of the Na⁺ current was more than twice that for the Na⁺ current found in retinal neurons. The glial Na⁺ current was half-inactivated at −55 mV. A transient component of the outward K⁺ current was blocked by external 4-aminopyridine while a more sustained component was blocked by external tetraethylammonium. At potentials between −150 and −50 mV the membrane behaved Ohmically. Voltage-gated currents in retinal astrocytes recorded in situ appear qualitatively similar to those described for some glial cells in vitro.     

Postsynaptic Currents and Short-term Synaptic Plasticity in Purkinje Cells Grafted onto an Uninjured Adult Cerebellar Cortex

It has been shown recently that embryonic Purkinje cells grafted extraparenchymally into an intact cerebellum, in the absence of any sign of damage, are able to migrate into the host molecular layer where they receive a climbing fibre innervation. Using the same technique, we investigated the development of the electrophysiological properties of the synapses between the grafted cells and their main afferents. Purkinje cells either in the graft or having migrated into the molecular layer of the host were recorded using the whole-cell patch-clamp method in acutely prepared slices 17–112 days after grafting. Spontaneous postsynaptic currents with a single-exponential decay and mediated by GABA_A receptors were very similar to those described in normal Purkinje cells. Excitatory postsynaptic currents (EPSCs) evoked by climbing fibre and by parallel fibre stimulation were blocked by an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate antagonist, and displayed the linear current-voltage relation typical of postnatal Purkinje cells. The attainment of normal functional properties by the adult axons at the newly formed synaptic sites was shown by the expression of short-term facilitation of parallel fibre EPSCs and of short-term depression of climbing fibre EPSCs. The grafted Purkinje cells showed climbing fibre polyinnervation 17-20 days after grafting which evolved to monoinnervation at 23-45 days, confirming the completion of the developmental programme up to maturation. Our experiments support the view that the adult intact brain is able to accept and integrate an additional number of neurons which show fully mature electrophysiological properties which are electrophysiologically indistinguishable from those of the host neurons.     

Sombati癫癎细胞模型电压依赖性钾电流的变化

目的：采用全细胞膜片钳记录技术研究Sombati癫癎细胞模型电压依赖性K＋电流的变化。方法：取SD新生乳鼠双侧海马,体外原代培养海马神经元,培养至第9天的部分海马神经元经无镁液处理3h制备癫癎细胞模型,采用全细胞膜片钳技术分别记录正常组（未经无镁液处理的海马神经元）和致癎组（经无镁液处理3h后更换为正常维持培养液的海马神经元）海马神经元的电压依赖性外向K＋电流。结果：致癎组外向K＋电流比正常组增大,峰值电流由（596.62±79.23）pA增至（978.68±53.23）pA,两组间比较差异有统计学意义（P=0.000）。与正常组相比,致癎组的I-V曲线明显左移,I-V曲线形态无显著改变。结论：Sombati癫癎细胞模型中,电压依赖性外向K＋电流显著增加可能是癫癎细胞模型癎性放电后为了保持细胞稳态的代偿性保护作用。     

Functional Properties of Neuronal Nicotinic Acetylcholine Receptor Channels Expressed in Transfected Human Cells

To study how subunit composition affects the functional properties of neuronal nicotinic acetylcholine receptors (nAChRs), we examined the behaviour of acetylcholine (ACh)-induced single-channel currents in human BOSC 23 cells transiently transfected with various subunit cDNA combinations. For all nAChRs examined (chick and rat α₃β₄, chick α_<3/β₂, α₄β₂, α₄β₄, α₇and α₈, expression levels were high enough to allow measurements of acetylcholine-evoked whole-cell currents and nicotine-elicited Ca²⁺ transients as well as the functional characterization of nAChR channels. Unitary acetylcholine-evoked events of α₈ nAChR had a slope conductance of 23 pS, whereas two conductance classes (19–23 and 32–45 pS) were identified for all other nAChR channels. The mean channel open times were significantly longer for homomeric α₇ and α₈ nAChRs (6–7 ms) than for heteromeric nAChRs (1–3 ms), with the exception of α₃α₄nAChRs (8.4 ms for rat, 7 ms for chick). At least two species of heterologously expressed nAChRs (α₃α₄and α₃α₂) exhibited single-channel characteristics similar to those reported for native receptors. The variety of nAChRs channel conductance and kinetic properties encountered in human cells transfected with nAChR subunits contributes to the functional diversity of nAChRs in nerve cells.     

Cyclic Nucleotide-gated Channels in Identified Human Olfactory Receptor Neurons

Patch-clamp recordings revealed the presence of a non-desensitizing cyclic nucleotide-gated channel on human olfactory receptor neurons and a fast-desensitizing non-specific cation channel activated by nucleotides on human supporting cells. Cyclic nucleotide-gated channels on olfactory receptor neurons showed selective channel activation by cAMP (K_1/2= 5 μM) and cGMP (K_1/2= 2 μM), a unitary conductance of ∼20 pS, a reversal potential of single-channel currents close to 0 mV, a linear current-voltage relationship over the range of −80 to 80 mV and a strong extracellular but a weaker intracellular blocking effect of Ca²⁺. The channel activity outlasted the cyclic nucleotide pulses for hundreds of milliseconds when higher agonist concentrations (>50 μM cAMP) were applied. The duration of the response was longer than in cyclic nucleotide-gated channels from other species studied so far. The plateau duration and the decay remained constant for pulses with a length of 50−150 ms, whereas pulses shorter than 50 ms successively reduced the time required by shortening the plateau phase. A larger difference for the K_1/2 values of cAMP (K_1/2= 22 μM) and cGMP (K_1/2= 2.5 μM) were found for a small group (n = 3) of cyclic nucleotide-gated channels, pointing to the selective expression of the a-subunit in a small subgroup of olfactory receptor neurons.     

Angiotensin II Induces Inward Currents in Subfornical Organ Neurones of Rats

The action of angiotensin II on subfornical organ (SFO) neurones was studied using whole-cell current and voltage-clamp recordings in rat slice preparations. In the current-clamp mode, membrane depolarization in response to angiotensin II was accompanied by an increased frequency of action potentials and an increased membrane conductance. In the voltage-clamp mode, angiotensin II elicited inward currents in a dose-dependent manner. The net angiotensin II-induced inward currents were voltage-independent, with a mean reversal potential of -29.8 +/- 6.2 mV. Amplitudes of the angiotensin II-induced inward currents were decreased during perfusion with a low sodium medium. The angiotensin II-induced inward currents were blocked by the AT1 antagonist losartan, and were partially blocked by the AT2 antagonist PD-123319. Neurones which were sensitive to angiotensin II were found in the peripheral region of the SFO, whereas neurones in the central region were less sensitive to angiotensin II. These results suggest that angiotensin II induces inward currents, with opening of nonselective cation channels through mainly AT1 receptors in a subpopulation of SFO neurones of rats.     

Currents evoked by GABA and glycine in acutely dissociated neurons from the rat medial preoptic nucleus

The responses of acutely dissociated medial preoptic neurons to application of GABA and glycine were studied using the perforated-patch whole-cell recording technique under voltage-clamp conditions. GABA, at a concentration of 1 mM, evoked outward currents in all cells (n=33) when studied at potentials positive to −80 mV. The I–V relation was roughly linear. The currents evoked by GABA were partially blocked by 25–75 μM picrotoxin and were also partially or completely blocked by 100–200 μM bicuculline. Glycine, at a concentration of 1 mM, did also evoke outward currents in all cells (n=12) when studied at potentials positive to −75 mV. The I–V relation was roughly linear. The currents evoked by glycine were largely blocked by 1 μM strychnine. In conclusion, the present work demonstrates that neurons from the medial preoptic nucleus of rat directly respond to the inhibitory transmitters GABA and glycine with currents that can be attributed to GABA_A receptors and glycine receptors respectively.     

Glycinergic Inhibitory Synaptic Currents and Related Receptor Channels in the Zebrafish Brain

To extend our study of the inhibitory synaptic network we have developed an isolated whole-brain preparation of the 52-h-old zebrafish (Brachydanio redo) in which the structural and functional integrity of the brain is preserved. We report the characterization of quantal inhibitory events and the correlation of their properties with those of the underlying activated channels. During whole-cell recordings of the Mauthner cells, applications of 10^?6 M tetrodotoxin greatly reduced the frequency and amplitude of the spontaneously occurring synaptic events, which were dominated by Cl^?-dependent inhibitory postsynaptic currents (IPSCs). Lowering Ca²⁺ and adding Mg²⁺ to tetrodotoxin-containing solutions resulted in a further decrease in amplitude of the recorded synaptic currents, the remaining ones being considered as miniature IPSCs (mISCs). Applications of 0.5–1 μMM strychnine in the presence of tetrodotoxin eliminated > 90% of the inhibitory currents in the preparation. The amplitude histograms of these mIPSCs exhibited two initial equally spaced peaks, followed by a skewed distribution for higher values. The first two components were well fitted by the sum of two Gaussian curves, giving a mean quantal amplitude of 35.7 pA (at a holding potential of-50 mV) and a coefficient of variation of 0.25 for the first peak. Outside-out recordings showed at least two classes of glycine receptor channels, one having multiple conductance levels with a main state of 81–86 pS and another displaying only one opening level of 41–43 pS. These two mean conductance states had similar mean open times, of 0.6–1 and 4.5–6 ms respectively. In addition, three mean closed times were observed for the 41–43 pS level. The shortest group (0.6–1 ms) was considered as representing gaps within bursts. Burst analysis revealed three mean burst durations, of 0.6, 4 and 35 ms. Comparisons of the amplitude of the first class of mIPSCs and of the open channel conductances indicated that one quantum opens 14–22 channels. Moreover, the correspondence between the mean decay time of mIPSCs and the mean open time or medium burst duration (4–5 ms) suggests that glycine-activated channels open only once in response to a single exocytosis. The pre-and postsynaptic origins of mIPSCs amplitude fluctuations are discussed in the context of multivesicular release versus the hypothesis of postsynaptic receptor saturation.     

Presenilins Upregulate Functional KChannel Currents in Mammalian Cells

Mutations in presenilin 1 (PS-1) and presenilin 2 (PS-2) have been linked to early onset, autosomal dominant Alzheimer's disease. Neither the normal function(s) of the presenilins nor their role(s) in mediating the devastating neurological and pathological changes associated with Alzheimer's Disease, however, are well understood. The results of the experiments described here demonstrate that expression of wild-type PS-1 or PS-2 increases outward K⁺current densities in HEK-293 cells relative to untransfected or mock-transfected cells. Western blot analysis reveals that there is a marked increase in full-length, rather than processed, presenilins in transiently transfected HEK-293 cells, suggesting that full-length PS-1 (or PS-2) underlies the observed increases in outward K⁺current densities. Consistent with this hypothesis, expression of an N-terminal proteolytic fragment of PS-1 is without effects on the membrane properties of HEK-293 cells. Mean outward K⁺current densities are also shown to be increased in HEK-293 cells expressing the exon 9 splice site PS-1 mutation (Δex9/PS-1), a mutant that does not undergo proteolytic processing. In HEK-293 cells transiently transfected with a missense (G209V) PS-1 mutant, however, mean K⁺current densities were not significantly different from controls. Expression of wild-type PS-1 in neonatal rat ventricular myocytes also results in increased outward K⁺currents, whereas no detectable effects on membrane currents were seen in PS-1-transfected COS-7 cells. These results suggest that the presenilins do not actually form K⁺channels, but rather that these proteins upregulate functional K⁺channel expression either directly by associating with K⁺channel pore-forming subunits or indirectly by increasing the synthesis, assembly, and/or transport of these subunits. The observation that PS-1 and PS-2 are highly expressed in neurons, localized to the endoplasmic reticulum, suggests that the presenilins could regulate neuronal K⁺channel expression; mutations in PS-1/PS-2 would then be expected to result in profound changes in neuronal excitability and contribute to the cognitive decline commonly associated with Alzheimer's Disease.     

Long-term Potentiation in the Striatum is Unmasked by Removing the Voltage-dependent Magnesium Block of NMDA Receptor Channels

We have studied the effects of tetanic stimulation of the corticostriatal pathway on the amplitude of striatal excitatory synaptic potentials. Recordings were obtained from a corticostriatal slice preparation by utilizing both extracellular and intracellular techniques. Under the control condition (1.2 mM external Mg2+), excitatory postsynaptic potentials (EPSPs) evoked by cortical stimulation were reversibly blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an antagonist of dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) ionotropic glutamate receptors, while they were not affected by 30 - 50 microM 2-amino-5-phosphonovalerate (APV), an antagonist of N-methyl-d-aspartate (NMDA) glutamate receptors. In the presence of 1.2 mM external Mg2+, tetanic activation of cortical inputs produced long-term depression (LTD) of both extracellularly and intracellularly recorded synaptic potentials. When Mg2+ was removed from the external medium, EPSP amplitude and duration increased. In Mg2+-free medium, cortically evoked EPSPs revealed an APV-sensitive component; in this condition tetanic stimulation produced long-term potentiation (LTP) of synaptic transmission. Incubation of the slices in 30 - 50 microM APV blocked striatal LTP, while it did not affect LTD. In Mg2+-free medium, incubation of the slices in 10 microM CNQX did not block the expression of striatal LTP. Intrinsic membrane properties (membrane potential, input resistance and firing pattern) of striatal neurons were altered neither by tetanic stimuli inducing LTD and LTP, nor by removal of Mg2+ from the external medium. These findings show that repetitive activation of cortical inputs can induce long-term changes of synaptic transmission in the striatum. Under control conditions NMDA receptor channels are inactivated by the voltage-dependent Mg2+ block and repetitive cortical stimulation induces LTD which does not require activation of NMDA channels. Removal of external Mg2+ deinactivates these channels and reveals a component of the EPSP which is potentiated by repetitive activation. Since the striatum has been involved in memory and in the storage of motor skills, LTD and LTP of synaptic transmission in this structure may provide the cellular substrate for motor learning and underlie the physiopathology of some movement disorders.     

Sex Differences in the Vomeronasal System

In the early eighties we found sex differences in the vomeronasal organ (VNO) and hypothesized that the vomeronasal system (VNS), a complex neural network involved in the control of reproductive behavior, might be sexually dimorphic. At that time sex differences had already been described for some structures that receive VNO input, such as the medial amygdala, the medial preoptic area, the ventromedial hypothalamic nucleus, and the ventral region of the premammillary nucleus. Since then, we have shown sex differences in the accessory olfactory bulb (AOB), the bed nucleus of the accessory olfactory tract (BAOT), and the bed nucleus of the stria terminalis (BST). When new VNS connections were found, all of them ended in nuclei that present sex differences. In general, sex differences in the olfactory system show two morphological patterns: one in which males present greater morphological measures than females, and just the opposite. To explain the morphometric measures of males in the latter, it has been hypothesized that androgens serve as inhibitors. Our work on the involvement of the GABA_A receptor in the development of AOB and maternal behavior sex differences also suggests that neonatal changes in neuronal membrane permeability to the ion Cl⁻ differences. This might be the first animal model to help us to understand the situation in which human genetic and gonadal sex do not agree with brain and behavioral sex. Finally, we stress that sex differences in the VNS constitute a neurofunctional model for understanding sex differences in reproductive behaviors.     

Localization in the Nervous System of Drosophila Melanogaster of a C-Terminus Anti-Peptide Antibody to a Cloned Drosophila Muscarinic Acetylcholine Receptor

Localization in the nervous system of Drosophila melanogaster of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) was investigated using a polyclonal antiserum raised against a peptide corresponding to the predicted receptor carboxyl terminal domain. Immunocytochemical studies on fly sections indicated that the product of the Dm1 mAChR gene was localized in the antennal lobes and in other regions of the brain and thoracic nervous system. Intense staining in the glomeruli of the antennal lobes, the region of the nervous system containing terminals of antennal olfactory sensory neurones and mechanosensory neurones, indicates possible roles for this mAChR gene product in the processing of olfactory and mechanosensory signals in the fly. The staining of a discrete group of neurosecretory cells in the pars intercerebralis of the brain indicates a possible new role for this mAChR in the regulation of neurosecretion. Very little staining is detected in the thoracic nervous system.     

Calcium-dependent inactivation of the monosynaptic NMDA EPSCs in rat hippocampal neurons in culture.

The effects of increased dendritic calcium concentration ([Ca2+]i) induced by single action potentials on monosynaptic glutamatergic excitatory postsynaptic currents (EPSCs) were studied in cultured rat hippocampal neurons. To investigate the respective roles of pre- and postsynaptic elements in the depolarization-induced NMDAR inactivation, we have performed simultaneous paired whole-cell recordings from monosynaptically connected pre- and postsynaptic hippocampal neurons. We report that the single firing of the postsynaptic neuron did not result in inactivation of the NMDAR-EPSC, whereas a burst of depolarizing steps transiently depressed the NMDAR-EPSCs in both pyramidal cells and interneurons. This effect was mediated by postsynaptic voltage-gated Ca2+ influx, as it was prevented by: (i) buffering postsynaptic [Ca2+]i with 30 mM BAPTA; (ii) removing extracellular Ca2+; or (iii) applying Cd2+o (100 microM), a voltage-gated calcium channel blocker. It does not involve presynaptic mechanisms as it selectively affected NMDA but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor-mediated EPSCs. These results suggest that inactivation of NMDAR-channels by voltage-gated Ca influx is a general property of hippocampal neurons, which may play an important role in reducing postsynaptic NMDAR Ca2+ influx that leads to plasticity or excitotoxicity during sustained neuronal activity.     

Methoxyverapamil Reduction of Nicotine-induced Catecholamine Release Involves Inhibition of Nicotinic Acetylcholine Receptor Currents

The mechanism by which the putative Ca²⁺ channel blocker methoxyverapamil (D600) inhibits nicotine-induced catecholamine release was investigated in bovine adrenal chromaffin cells and in neurons from paravertebral sympathetic ganglia of chick embryos. We found D600 to prevent catecholamine release evoked by 30 s applications of nicotine with a significantly higher potency than the release induced either by 30 s K⁺ depolarizations or by electrical field stimulation of sympathetic neurons. Like the use-dependent action of D600 upon Ca²⁺ channels, the magnitude of inhibition of the K⁺-evoked secretion depended on the duration of stimulation (10 s to 5 min). Data on catecholamine release were supplemented by patch-clamp recordings. We found whole-cell currents in chromaffin cells evoked by (extrapolated) 0.5 s applications of nicotine to be significantly more sensitive to D600 than Ca²⁺ currents induced by a 0.5 s depolarization from -80 to 0 mV. In both instances, the potency of D600 depended on the duration of the (nicotinic and depolarizing) stimuli. Our data suggest that D600 inhibits nicotine-induced catecholamine release by reducing nicotinic acetylcholine receptor currents rather than voltage-gated Ca²⁺ currents. Hence, in chromaffin cells as well as in sympathetic neuronal preparations, D600 does not appear to be a suitable tool to investigate the part voltage-activated Ca²⁺ currents play in cellular events induced by nicotine.     

Androgenic and Oestrogenic Influences on Tyrosine Hydroxylase‐Immunoreactive Cells of the Prairie Vole Medial Amygdala and Bed Nucleus of the Stria Terminalis

The posterodorsal medial amygdala (MeApd) and principal nucleus of the bed nucleus of the stria terminalis (pBST) are densely interconnected sites integrating steroid hormone and olfactory information necessary for sociosexual behaviours in many rodents. Our laboratory recently reported sexually dimorphic populations of cells containing tyrosine hydroxylase (TH) located in the MeApd and pBST of prairie voles (Microtus ochrogaster), with males having many more TH‐immunoreactive (‐ir) cells in these sites than do females. Gonadal hormones circulating during adulthood were showm to regulate this sex difference because it was eliminated by castrating adult males or implanting females with testosterone‐filled capsules. In the present study, we demonstrate that many (25–65%) TH‐ir cells in the MeApd and pBST of adult virgin male and female prairie voles also contain immunoreactivity for either the androgen receptor or oestrogen receptor α. Subcutaneous implants of oestradiol benzoate mimicked the effects of testosterone and maintained high numbers of TH‐ir cells in these sites in castrated males. However, implants of dihydrotestosterone (DHT) did not, and these males had low numbers of TH‐ir cells similar to castrated males given empty capsules. A similar effect was found in females, where testosterone or oestradiol benzoate greatly increased the number of TH‐ir cells in these sites compared to intact or ovariectomised controls, but DHT did not. DHT implants did, however, maintain high seminal vesicle weights in males. Thus, many of the TH‐ir cells in the prairie vole MeApd and pBST are potentially sensitive to androgens and oestrogens, although maintaining immunocytochemically detectable levels of TH in these cells may depend more on an oestrogen‐mediated mechanism in both sexes. These data have implications for understanding how gonadal hormone release across the reproductive cycle modulates these species‐specific groups of catecholaminergic cells and socially monogamous behaviours in prairie voles.     

Photoreceptor Cells of the Pike Pineal Organ as Cellular Circadian Oscillators

In the pike pineal, the rhythm of melatonin (MEL) secretion is driven by a population of cellular circadian oscillators, synchronized by the 24 h light/dark (LD) cycle. Because the pineal photoreceptor contains both the input and output pathways of the clock, this cell is likely to be a cellular circadian system by itself. To support this idea, we have dissociated and cultured pike pineal cells as well as purified photoreceptors. In culture, the pineal cells reassociated in follicles, surrounded by collagen fibres. At the electron microscopic level, they appeared well preserved. Total cells consisted mainly of photoreceptors and glia. Purified cells corresponded exclusively to photoreceptors. Under LD, MEL production was rhythmic. Under constant darkness (DD), the rhythm was well sustained for at least six 24 h cycles (τ= 24/27 h) with 1 × 10⁶ total cells/well or below; with 2 × 10⁶ total cells/well, a strong damping occurred towards high levels as soon as after the second cycle. At the density of 0.5 × 10⁶ cells/well, purified photoreceptors produced less MEL than an equivalent amount of total cells. However, the pattern of the oscillations was similar to that observed with 2 × 10⁶ total cells, i.e. a damping occurred rapidly. Decreasing the density to 0.125 × 10⁶ photoreceptors/well resulted in a loss of homogeneity among replicates. The production of melatonin by single photoreceptors was monitored by means of the reverse haemolytic plaque assay. Both under LD and under DD, the number of photoreceptors releasing melatonin was higher during the (subjective) dark than during the (subjective) light. The results provide strong support to the idea that the pike pineal photoreceptor is a cellular circadian system. Expression of the oscillations seemed to depend on several factors, including cell to cell contacts between photoreceptors. There is indication that also MEL and glia might be involved.