NF-κB subunits are differentially distributed in cells of lumbar dorsal root ganglia in naïve and diabetic rats.

Previous studies demonstrated that nuclear factor κB (NF-κB) activation is decreased in dorsal root ganglia (DRG) of rats having streptozotocin (STZ)-induced diabetes. DRG contain cell bodies of neurons that convey sensory signals from the periphery. To determine the relationship between diabetes-induced neuropathy and NF-κB expression in DRG, behavioral, immunohistochemical, and biochemical studies were performed on naïve and 3-month diabetic rats. Behavioral studies confirmed that many diabetic rats develop tactile allodynia, or increased sensitivity to light touch, in the hind paws. Immunohistochemical studies on lumbar DRG that receive input from the affected regions revealed that p50 and p65, frequent NF-κB subunit partners, are differentially localized. Intense p65 immunostaining was detected in the cytoplasm of small- and medium-sized neurons as well as in satellite cells. In contrast, p50 was localized in the cytoplasm of virtually all neurons. In many cases, prominent staining was also present in nuclei, a location consistent with transcription factor activation. Immunohistochemical and biochemical studies found that the nuclear to cytoplasmic ratio of p50 expression was significantly reduced in diabetic rats compared to that in naïve animals. Our findings raise the possibility that changes in NF-κB activation in a subset of DRG neurons participates in mediating diabetes-induced sensory neuropathy.

Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression.

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.

Neuregulin induces GABA(A) receptor subunit expression and neurite outgrowth in cerebellar granule cells.

Neuregulin (NRG), a growth and differentiation factor that signals via erbB receptor tyrosine kinases, has been shown to have biological effects in both the CNS and the peripheral nervous system. We report here that erbB4 is expressed in mature cerebellar granule cells, where it appears to be concentrated at the granule cell postsynaptic terminals. We also show that one form of NRG, Ig-NRG, plays a crucial role in aspects of cerebellar granule cell development in vitro. First, Ig-NRG treatment of granule cells in culture selectively induces the expression of the GABA(A) receptor beta2 subunit. This increase in subunit expression is paralleled by an increase in functional GABA(A) receptors. In contrast to its effects on GABA(A) receptor subunit expression, Ig-NRG does not upregulate NMDA receptor N2B and N2C subunit expression. Second, we demonstrate that Ig-NRG also enhances neurite outgrowth from cultured granule cells. Ig-NRG does not, however, act as a survival factor for the granule cells. We have compared the effect of Ig-NRG with the effects of brain-derived neurotrophic factor (BDNF), a neurotrophin that exerts specific effects on granule cells in culture, and found that BDNF does not mimic the effects of Ig-NRG on GABA(A) receptor subunit expression. Our results show that Ig-NRG has specific effects on granule cell development and maturation and may regulate these processes in vivo.

Strategies for early discharge of the hospitalized patient with community-acquired pneumonia.

The treatment of the hospitalized patient with uncomplicated CAP is changing, to include a brief period of intravenous antibiotics followed by oral therapy. The Classification of Community-Acquired Pneumonia or CoCAP is a stratification tool that categorizes patients as low-risk pneumonia, unstable pneumonia, or complicated pneumonia. Use of validated hospital admission criteria, combined with the CoCAP algorithm and evolving criteria for switching patients from intravenous to oral therapy provides a structure for organizing treatment of patients with CAP for caregivers. Patients who can be discharged early are those from the unstable pneumonia group, which includes patients who have had reversal of their metabolic problems and stabilization of comorbid conditions, and who have not developed any serious pneumonia-related complications. Prolonged courses of intravenous antibiotic therapy are being replaced with 2 to 3 day courses of intravenous hydration and antibiotics; a switch to oral therapy and hospital discharge can be achieved after the patient tolerates one dose of oral therapy. Parameters to watch include vital signs and white blood cell count. Provided these parameters are improving, although they may not have returned to normal, the patient can be switched to oral therapy. Although patient treatment guidelines and critical pathways are becoming widespread in disease management, CAP is one disease in which prospective studies have demonstrated that a reduction in hospital stay is safe. Patients, caregivers, and administrators are happy with the reduction in hospital LOS. Other treatment protocols are being explored, including a single dose of intravenous antibiotic prior to oral switch and all-oral regimens employing the newer fluoroquinolones.

Immature granule neurons from cerebella of different ages exhibit distinct developmental potentials.

Cultured cerebellar granule neurons exhibit different developmental potentials in culture dependent on cerebellar age at plating. In cultures prepared at postnatal days (P)2-6, when all granule neurons reside in the external germinal layer (EGL) in vivo, levels of the GABA(A) receptor beta2 and gamma2 subunit mRNAs are constant. In contrast, in cultures prepared at P8-10, when neurons have begun to migrate into the internal granule cell layer (IGL), the mRNAs increase several-fold in a pattern mimicking that found in vivo. To determine the relationship between neuronal differentiation in culture and potential to express GABA(A) receptor beta2 and gamma2 subunit transcripts in the mature pattern, neuronal maturity in P6 and P10 cultures was compared. Bromodeoxyuridine labeling studies demonstrated that P10 as well as P6 cultures contained neurons only from the EGL. Moreover, the maturation of cultured P10 and P6 neurons appeared virtually identical. Cells dissociated at both ages expressed mRNAs encoding the EGL markers MATH-1 and TAG-1. The MATH-1 transcript disappeared from cultures maintained 4 days when expression of the GABA(A) receptor alpha6 subunit, a marker of mature cells, was initiated. Thus, although cultured P6 and P10 granule neurons exhibit the same maturation markers, P10 neurons presumably have been modulated by environmental cues specifying increases in GABA(A) receptor beta2 and gamma2 subunit expression. This possibility is supported by the finding that extracts of dissociated P10 cells but not P6 cells induce increases in GABA(A) receptor subunit expression in P6 cultures.

Comparison of 7 versus 10 days of antibiotic therapy for hospitalized patients with uncomplicated community-acquired pneumonia: a prospective, randomized, double-blind study.

The objective of this study was to compare the outcome of 7 versus 10 days of antibiotic therapy for inpatients with moderately severe community-acquired pneumonia (CAP). A prospective, randomized, double-blind study with a follow-up period of 42 days was conducted. Fifty-two veterans were treated with 2 days of cefuroxime at 750 mg intravenously every 8 hours followed by group 1, 8 days oral therapy, and group 2, 5 days oral therapy followed by 3 days of placebo. Oral therapy consisted of cefuroxime axetil at 500 mg every 12 hours. No difference was seen in cure rates: 20 of 22 (90.9%) patients in group 1 and 21 of 24 (87.5%) patients in group 2. There were no late recurrences. Potential US cost-savings is $27.2 million. Inpatients with moderately severe CAP can be treated with 2 days of intravenous antibiotics followed by 5 additional days of oral antibiotics. Longer treatment duration prolongs the cost of care, without increasing the cure rate or decreasing the pneumonia recurrence rate.

Developmental expression of cerebellar GABAA-receptor subunit mRNAs. Nature versus nurture.

Recent studies have demonstrated that many of the mRNAs encoding GABAA-receptor subunits in the cerebellum exhibit distinct temporal profiles of expression. The levels of six of these subunit transcripts increase severalfold in the second week of postnatal ontogeny. Findings from a variety of experimental systems suggest that the onset and increases in subunit mRNA expression are mediated by the interaction of genetic and epigenetic programs. The initiation of subunit mRNA expression occurs relatively early in cellular maturation and may be directed by intrinsic mechanisms. However, the levels of expression attained in adult animals may be controlled by extrinsic signals received by neurons during the postnatal maturation process.

NMDA receptor stimulation selectively initiates GABA(A) receptor delta subunit mRNA expression in cultured rat cerebellar granule neurons.

Findings in vivo and in culture suggest that neuronal activity selectively regulates GABA(A) receptor delta subunit mRNA expression in cerebellar granule neurons. For example, the onset of delta subunit mRNA expression during postnatal maturation coincides with innervation. Furthermore, depolarizing conditions (25 mM KCl) in culture initiate and maintain increases in the delta subunit transcript level. We have now examined whether similar changes in delta subunit mRNA expression occur in cultured neurons after activation of glutamate receptors of the NMDA subtype, an event that mimics granule neuron depolarization by mossy fiber innervation in vivo. Our studies demonstrate that addition of 50 microM NMDA to cultured rat granule neurons maintained in defined, serum-free medium specifically initiates delta subunit transcript expression. Whereas the level of the delta subunit mRNA is increased fourfold by this treatment, levels of other GABA(A) receptor subunit transcripts are not significantly changed. The level of the delta subunit transcript is further increased when NMDA receptor activation is enhanced by maintaining neurons in a Mg2+-free medium to alleviate Mg2+ blockade of the receptor channel. The NMDA-induced elevation in delta subunit transcript expression involves activation of a Ca2+/calmodulin-dependent protein kinase pathway. These findings suggest that activation of an excitatory pathway may regulate the expression of an inhibitory receptor phenotype in cerebellar granule neurons in vivo.

Expression of the GABAA receptor delta subunit is selectively modulated by depolarization in cultured rat cerebellar granule neurons.

The levels of several GABAA receptor subunit mRNAs increase as cerebellar granule neurons migrate to their adult positions and receive excitatory mossy fiber input. Despite the temporal similarity of these increases in transcript expression in vivo, studies in cultured granule neurons demonstrated that the subunit mRNAs are differentially regulated. To address the possibility that neuronal activity regulates transcript expression, GABAA receptor subunit mRNA levels were assessed in cultured granule neurons grown in chemically defined, serum-free medium containing either nondepolarizing (5 mM) or depolarizing (25 mM) KCl concentrations. Whereas the delta subunit mRNA was almost undetectable in cultures maintained in nondepolarizing medium, an eightfold increase occurred between days 2 and 4 in cultures grown in depolarizing medium. Furthermore, delta subunit transcript expression was reduced by 76 +/- 6% when neurons in depolarizing medium were switched into nondepolarizing medium. The importance of depolarization in the initiation and maintenance of subunit transcript expression in granule neurons was selective for the GABAA receptor delta subunit. These changes in transcript expression involved calcium entry through L-type calcium channels. Nifedipine treatment (1 microM) both reduced intracellular calcium and decreased delta subunit mRNA expression by 79 +/- 4%. Furthermore, inhibition of Ca2+/calmodulin-dependent protein kinases (CaM kinases) by KN-62 (1 microM) also reduced delta subunit transcript expression. These studies demonstrate that KCl-induced depolarization, a condition that mimics the effects of neuronal activity, selectively modulates GABAA receptor delta subunit mRNA expression through a pathway involving calcium entry and activation of a CaM kinase.

GABAA receptor subunit expression and assembly in cultured rat cerebellar granule neurons.

The assembly of multisubunit GABAA receptors in specific neuronal populations is a complex process which is poorly understood. To begin to examine receptor assembly, alpha 1, beta 2/3, and gamma 2 subunit polypeptide expression and association, as well as receptor binding, were examined in cultured rat cerebellar granule neurons. Western blots revealed two alpha 1-immunoreactive proteins. A 39 kDa species was maximal at 2 days in culture and subsequently declined. In contrast, a 51 kDa polypeptide, the anticipated size of the mature alpha 1 subunit, was first detected at 4 days and increased throughout the culture period. Additional studies demonstrated that the beta 2/3 and gamma 2 subunits were detectable at 2 days and attained maximal levels by 6 days. The level of [3H]Ro15-1788 binding, a measure of assembled receptors, rose in parallel with the increases in the 51 kDa alpha 1, beta 2/3 and gamma 2 subunits. Moreover, the 51 kDa alpha 1, beta 2/3, and gamma 2 subunits were associated in receptor complexes. However, immunohistochemical studies demonstrated the presence of substantial intracellular subunit staining. This finding suggest that only some of the subunits expressed in granule neurons contribute to functional GABAA receptors on the cell surface.

A prospective randomized study of inpatient iv. antibiotics for community-acquired pneumonia. The optimal duration of therapy.

STUDY OBJECTIVE: To compare therapeutic outcome and perform a cost-benefit analysis of inpatients with community-acquired pneumonia (CAP) treated with a shortened course of i.v. antibiotic therapy. DESIGN: A prospective, randomized, parallel group study with a follow-up period of 28 days. SETTING: Bronx Veterans Affairs Medical Center (VAMC) and the Castle Point VAMC; university-affiliated VAMC general medical wards from September 1993 to March 1995. PATIENTS: Seventy-two male veterans and 1 female veteran with 75 episodes of CAP defined by a new infiltrate on chest radiograph and either history or physical findings consistent with pneumonia. Study population was 42%(31) black, 33%(24) white, and 25%(18) Hispanic. INTERVENTIONS: Patients were randomized (1:1:1) to 1 of 3 treatment groups: group 1 received 2 days of i.v. and 8 days of oral therapy; group 2 received 5 days of i.v. and 5 days of oral therapy; and group 3 received 10 days of i.v. therapy. Antibiotics consisted of cefuroxime, 750 mg every 8 h for the i.v. course, and cefuroxime axetil, 500 mg every 12 h for the oral therapy. MEASUREMENTS AND RESULTS: No differences were found in the clinical course, cure rates, or resolution of chest radiograph abnormalities among the three groups. A significant difference was found in the length of stay (LOS) among the three groups. The mean +/- SD LOS was 6 +/- 3 days in group 1, 8 +/- 2 days in group 2, and 11 +/- 1 days in group 3. The shortened LOS could potentially save $95.5 million for the Department of Veterans Affairs and $2.9 billion for the US private sector. CONCLUSIONS: Adult patients hospitalized for CAP who are not severely ill can be successfully treated with an abbreviated (2-day) course of i.v. antibiotics and then switched to oral therapy. A longer course of i.v. therapy prolongs hospital stay and cost, without improving the therapeutic cure rate.

Differential regulation of GABA A receptor subunit mRNAs in rat cerebellar granule neurons: importance of environmental cues.

Levels of the GABA A receptor alpha1-, alpha6-, beta2-, beta3-, gamma2-, and delta-subunit mRNAs in cerebellar granule neurons rise concurrently during the second week of postnatal ontogeny. Previous studies in culture have suggested that extrinsic signals control these increases, but little is known about the nature of the regulatory cues. To determine when granule neurons become competent to express these six subunit mRNAs in mature patterns and to gain insight into their regulation, reverse transcriptase-PCR was used to examine transcript expression in cultured granule neurons prepared at 2-day intervals from postnatal days 2 through 10. Although only one pattern of expression was observed in vivo, three patterns were detected in culture. First, the levels of the alpha1- and alpha6-subunit mRNAs were constant in cultures prepared at all ages. Second, the levels of the beta2-, beta3-, and gamma2-subunit mRNAs were constant in cultures prepared at postnatal days 2-6 but increased in those prepared at days 8-10. Third, the delta-subunit mRNA level increased over time in culture regardless of cerebellar age at plating. Moreover, only delta-subunit transcript expression was modulated by cell density. These findings indicate that the subunit transcripts are differentially regulated by multiple environmental cues.

GABAA receptor subunit mRNA expression in the weaver cerebellum: modulation by cell-cell interactions.

Recent studies have suggested that the developmental expression of GABA(A) receptor subunit mRNAs in cerebellar Purkinje neurons is modulated by cell--cell interactions [correction of interacactions]. In this population, the levels of mRNAs encoding the alpha1, beta2, and gamma2 subunits increase simultaneously during the second week of postnatal ontogeny, a period temporally coincident with cerebellar maturation and synapse formation. To determine the importance of cell--cell interactions in modulating receptor gene expression, the levels of GABA(A) receptor subunit mRNAs in Purkinje neurons of weaver mice and littermate controls were examined by quantitative in situ hybridization histochemistry. In the weaver mutant most granule neurons die early in postnatal development, thus eliminating the major source of excitatory input to Purkinje cells. Despite this loss, the three subunit mRNAs were expressed in all Purkinje neurons. However, the levels of expression were generally lower in the mutants than in the littermate controls. These results suggest that the onset of GABA(A) receptor gene expression in cerebellar Purkinje neurons occurs in the absence of extensive synapse formation by mechanisms which may be intrinsic to the neurons. In contrast, the absolute level of transcript expression attained appears to be modulated by cell-cell interactions or by other extrinsic cues present in the cerebellar environment.

GABAA receptor subunit polypeptides increase in parallel but exhibit distinct distributions in the developing rat cerebellum.

The GABAA receptor, a multisubunit ligand-gated ion channel, plays a central role in cell-cell communication in the developing and adult nervous system. Although the developmental expression of mRNAs encoding many subunit isoforms has been extensively characterized throughout the central nervous system, little is known concerning the relationship between subunit mRNA and polypeptide expression. To address this issue, we examined the developmental expression of the alpha 1, beta 2/3, and gamma 2 subunit polypeptides, subunits that are thought to coassemble in many brain regions. Western blot analysis using subunit-specific antibodies revealed that the levels of these polypeptides in both the cerebral cortex and cerebellum increased severalfold during the second postnatal week. Whereas polypeptide expression in the cerebellum paralleled that of the corresponding subunit mRNAs, increases in beta 2/3 and gamma 2 polypeptide expression in the cerebral cortex occurred in the absence of detectable changes in the mRNA levels. To determine whether the increases in subunit polypeptide expression in the cerebellum were accompanied by changes in distribution, immunohistochemistry was performed. These studies demonstrated that the subunits exhibited different but partially overlapping distributions that remained constant throughout postnatal development. Our findings suggest that although GABAA receptor subunit polypeptide expression may be regulated primarily at the level of the mRNA, additional regulatory mechanisms may play a role. Furthermore, the observation that subunit distribution remains constant in the cell bodies of cerebellar Purkinje neurons, which express the alpha 1, beta 2, beta 3, and gamma 2 subunit mRNAs exclusively, suggests that GABAA receptor subunit composition in this cell population does not change during postnatal maturation.

Galanin and vasoactive intestinal peptide messenger RNAs increase following axotomy of adult sympathetic neurons.

The adult rat superior cervical ganglion (SCG) contains low levels of galanin- and vasoactive intestinal peptide-(VIP) like immunoreactivity, with very few immunostained principal neurons. Immunoreactivity for both neuropeptides increases in these neurons after explantation or postganglionic axotomy in vivo. Northern blot analysis has demonstrated concomitant increases in mRNAs encoding these peptides. To localize cells in axotomized ganglia which increase their expression of these mRNAs, we performed in situ hybridization studies. In control SCG, only a few principal neurons contained mRNA for either galanin or VIP. After 48 h in organ culture, galanin mRNA was expressed in the majority of principal neurons. At 48 h after in vivo axotomy of both postganglionic trunks of the SCG, the internal and external carotid nerves, the distribution and number of neurons, expressing galanin mRNA increased similarly to that seen in culture. Lesioning either trunk alone produced increases in galanin mRNA localized to those regions of the ganglion containing neurons that project into the lesioned trunk. Transection of the predominantly preganglionic cervical sympathetic trunk increased galanin mRNA expression in a small population of neurons near that nerve trunk. The distributions of these labeled neurons, together with previous neuroanatomical studies, suggests that they had been axotomized by the lesions. Similar studies examining VIP mRNA expression demonstrated that although considerably fewer VIP mRNA expressing neurons than galanin mRNA expressing neurons were present after axotomy, the distribution of neuropeptide mRNA-positive cells were similar in both cases. These observations suggest that increases in the peptides galanin and VIP after nerve transection result from changes in the levels of their mRNAs in those neurons that have been axotomized.

Rapid decline of GABAA receptor subunit mRNA expression in hippocampus following transient cerebral ischemia in the gerbil.

Inhibitory neurotransmission may play an important role in neuronal degeneration following transient cerebral ischemia. We studied the effect of transient forebrain ischemia on the GABAA receptor system in the gerbil hippocampus. Gerbils were subjected to 5 minutes of bilateral carotid occlusion and were sacrificed at various times over 4 days following reperfusion. There was a substantial loss of pyramidal cells in the CA1 area of the hippocampus 4 days following ischemia. No cell loss was detected in CA3 pyramidal cells of the hippocampus, granule cell layer of the dentate gyrus, and ventroposterior medial and ventroposterior lateral nuclei of the thalamus at any time following ischemia. Examination of brain slices by in situ hybridization histochemistry revealed that a change in expression of the GABAA receptor alpha 1 and beta 2 subunit mRNAs occurred in two phases following onset of reperfusion. The early phase (rapid) occurred within the first 4 hours following reperfusion. The expression of mRNAs significantly decreased (up to 25%) within 1 hour after occlusion in CA1 and CA3 pyramidal cell layers of the hippocampus and in the granule cell layer of the dentate gyrus. The expression of the mRNAs in these regions continued to decrease for 4 hours (up to 43%). In the second phase, which began between 4 and 12 hours following reperfusion, mRNA expression started to return to control levels in CA3 hippocampus and in the dentate. However, expression of both mRNAs continued to decline slowly in the CA1 pyramidal cell layer (up to 85%) over the next 3 days, concomitantly with degeneration of the CA1 pyramidal cells. Expression of mRNAs in the ventroposterior medial or ventroposterior lateral nuclei of the thalamus was similar to control values. To determine if a change in GABAA receptor distribution paralleled changes in receptor subunit mRNA expression, we also measured the binding of [35S]t-butylbicyclophosphorothionate to GABAA receptor chloride channels. The t-butylbicyclophosphorothionate [35S] binding decreased between 1 and 4 days after reperfusion in the dendritic fields of CA1 pyramidal cells (strata oriens, radiatum, and lacunosum-moleculare) but not in the pyramidal cell body layer. These results indicate that expression of GABAA receptor subunit mRNAs decrease well before CA1 pyramidal cell degeneration and loss of GABAA receptors. At present, it is not clear if an early loss of mRNA expression after an ischemic insult leads to a functional defect in GABAA receptors. If so, a loss of GABA neurotransmission may contribute to the development of neuronal degeneration following cerebral ischemia.(ABSTRACT TRUNCATED AT 400 WORDS)